WO1995035369A1 - METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS - Google Patents

METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS Download PDF

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WO1995035369A1
WO1995035369A1 PCT/US1995/007968 US9507968W WO9535369A1 WO 1995035369 A1 WO1995035369 A1 WO 1995035369A1 US 9507968 W US9507968 W US 9507968W WO 9535369 A1 WO9535369 A1 WO 9535369A1
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molecule
region
primer
reverse transcriptase
template
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PCT/US1995/007968
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WO1995035369A9 (en
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Jeffrey E. Miller
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Miller Jeffrey E
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Priority to JP8502600A priority Critical patent/JPH10501974A/en
Priority to EP95926600A priority patent/EP0781330A1/en
Priority to AU30921/95A priority patent/AU3092195A/en
Publication of WO1995035369A1 publication Critical patent/WO1995035369A1/en
Publication of WO1995035369A9 publication Critical patent/WO1995035369A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the present invention relates to methods and compositions for DNA synthesis, and, more particularly, for the synthesis of complementary DNA.
  • the present invention is a tool for molecular biology.
  • An introduction to the nomenclature of molecular biology, the structure of DNA, RNA and proteins and the interrelationships between these molecules, is provided in Chapter 4, Synthesis of Proteins and Nucleic Acids of Darnell et al . , Molecular Cell Biology. Scientific American Books (1989) .
  • a more detailed treatment of these issues is set forth in the full text of Darnell et al . , (1989) and in Lewin, Genes IV. Oxford University Press (1990) .
  • Hereditary information is encoded in the genes of an organism. Genes are composed of polymers of nucleic acids. In higher organisms this nucleic acid is deoxyribonucleic acid (DNA) . DNA is composed of a series of four nucleotide bases; the hereditary information carried by a gene is encoded by the specific sequence of nucleotide bases in the DNA molecule.
  • the genetic information within structural genes encodes proteins; the sequence and structure (and therefore function) of a particular protein is determined by the order of the nucleotide bases within the gene that encodes that protein. Proteins determine an organism's identity; from cellular structures to the organism's response to its environment. Thus, the genes that encode these proteins determine an organism's identity.
  • messenger RNA messenger RNA
  • RNA messenger RNA
  • genes are made up of coding regions (termed “exons”) and non-coding regions (termed “introns”) .
  • Exons directly encode the protein sequence of the gene.
  • Introns may be very large and there may be a large number of intron sequences within a particular gene. The role of the non-coding intron sequences is unclear.
  • these intervening sequences serve at least three critical purposes: first, they allow a cell to produce a nascent RNA transcript which can be spliced in several ways to generate a number of different proteins; second, they allow the inclusion of control elements within the intron regions which enhance the regulation of gene expression; and three, they relegate discrete portions or cassettes of protein sequence to exon units which can be more easily shuffled during the course of evolution and therefore facilitate the development of new proteins which may ultimately enhance the survival of the species.
  • the transcription process involves the formation of an mRNA copy of the entire gene. That is, the mRNA produced by the transcription process contains a copy of both the non-coding intron sequences and the protein- encoding exon sequences.
  • the mRNA first produced by transcription is the same length as the gene from which it was copied. Subsequently, this immature mRNA undergoes a processing stage during which the non-coding intron sequences are spliced out.
  • the resulting processed mRNA molecules thus contain only the information required to encode the protein (i.e. they contain copies of only the joined exon sequences) .
  • These processed mRNA molecules are thus considerably shorter in length than the "genomic sequence" (the gene exons and introns as they exist in the chromosome) from which the mRNA was initially copied.
  • the processed mRNA is also modified at this stage to include a polyriboadenylic acid, poly(A) , tail at one end of the molecule (the 3'- end) and a "cap" structure at the other end of the molecule (the 5'-end) (standard nomenclature assigns one end of DNA and RNA molecules as the 5'-end and the other as the 3'-end, according to the terminal chemical groupings of the molecule) .
  • An mRNA molecule that has been processed to remove introns and has a 5'-cap and a 3'-poly(A) tail is termed a "mature" mRNA molecule.
  • a greatly simplified diagram of the transcription process, illustrating removal of the non-coding intron sequences is shown in Figure 1.
  • translation The step of converting the information carried by the mature messenger RNA into a protein is termed translation.
  • Translation is the final step of the means by which the information encoded by the nucleotide sequence within a structural gene is converted into a specific protein composed of a sequence of amino acids.
  • the size of a gene is measured in the number of nucleotide bases that it contains, usually expressed in terms of thousands of bases (kilobases or Kb) . Although there are several examples of larger genes, the total coding sequence of most structural genes (the exons) typically totals 1-10 Kb. However, the presence of multiple large intron sequences between the exon segments means that at the genomic level these genes are spread out over a much larger area, frequently spanning tens or even hundreds of kilobases.
  • YACs Yeast Artificial Chromosomes
  • YACs Yeast Artificial Chromosomes
  • the gene contains many intron sequences, and is thus large, years of effort can be expended in attempting to recover the remaining pieces of the gene. Additional effort may then be required to determine the relative order of the gene fragments and to distinguish exon from intron sequences.
  • the ability to clone a gene as a contiguous protein coding cassette is particularly important where identification of the gene is achieved by means of a detection technique which relies on production of the protein in a recombinant bacterial or viral system and "screening" for the function or structure of the desired protein — a common technique of detecting cloned genes.
  • cDNA Complementary DNA
  • Bacteria are commonly used as hosts in which a cloned gene is expressed.
  • the genes of prokaryotes, including bacteria, do not contain introns, and so these cells do not have the splicing machinery necessary to process immature mRNA into a mature mRNA that can be translated into a functional protein.
  • Genomic clones of eukaryotic genes i.e., containing introns and exons
  • can not be expressed in a bacterial host whereas a cDNA copy of the same gene can be expressed - either in prokaryotes or eukaryotes.
  • cDNA clones are routinely used for large scale protein production. This artificial protein expression is termed "recombinant protein" production and is an increasingly common way of producing many of the pharmaceuticals which for years were accessible in small amounts by tedious extraction from other animal's tissues.
  • RNAse Ribonuclease enzymes are very stable and so even a very small amount of the active enzyme in an mRNA preparation will cause problems. RNAse is present on virtually all surfaces, including human skin, and is thus very easily introduced into the RNA preparation. To avoid contamination problems, all solutions, glassware and plasticware must be specially treated.
  • RNA degradation starts at the first step of breaking open the cells (the cells themselves contain ribonucleases which, upon lysis of the cells, come into contact with the RNA) , and continue throughout the procedure.
  • Total RNA extracted from a cell is made up of messenger RNA (mRNA) , transfer RNA (tRNA) and ribosomal RNA (rRNA) .
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • the mRNA typically makes up only 1-3% of the total cellular RNA (approximately 1 x I0 "1 g mRNA per eukaryotic cell, Sargent, T.D., Methods Enzymol . 152:423- 432 (1987)).
  • Most cDNA synthesis reactions rely on the presence of the poly(A) tail present only in mature mRNA transcripts.
  • the mature RNA transcripts are selectively extracted from the bulk of the cellular RNA, usually by affinity chromatography. This is an essential step for successful in vitro cDNA synthesis; failure to enrich for the mature mRNA frequently results in a low yield of poor quality cDNA.
  • the mRNA preparation may be size selected. This is usually performed to remove the smaller size molecules (usually degraded forms of larger mRNAs) which would otherwise interfere with the cDNA cloning procedure. Size selection may also be performed to enrich for an mRNA species of known size. Size selection may be performed by electrophoresis through agarose gels, by column chromatography, or by sedimentation through sucrose gradients. These techniques result in lower yields, and may require the presence of methylmercuric hydroxide to disrupt secondary intramolecular structure. Methylmercuric hydroxide is extremely toxic and volatile, and requires great care in handling. Safer alternatives (such as the use of gels containing glyoxal/dimethyl sulfoxide or formaldehyde) are available, but these techniques also involve dangerous chemicals and have associated disadvantages.
  • cDNA cloning is often used to assess which genes are expressed in a cell under particular conditions or at a particular stage in the development of the organism.
  • the time and conditions required to extract the mRNA may themselves produce alterations in the gene expression pattern of the cell.
  • mRNA molecules which are present in very low abundance (estimated at 20 copies per cell) or which are unstable may be lost during the RNA isolation procedure.
  • cDNA synthesis is performed in vitro. All methodologies presently used for cDNA synthesis follow mRNA extraction and purification, or are performed in situ or under in vitro conditions. These methodologies are reviewed in detail in Kimmel and Berger (1987); Okayama, H. , et al . , (1987) ; Van Gelder, et al . , Proc. Natl . Acad. Sci . USA 87:1663-1667 (1990); and Embleton, M.J., et al . , Nucleic Acids Res. 20:3831-3837 (1992)). All of the presently available techniques utilize RNA-dependent DNA polymerase enzymes (more commonly termed reverse transcriptase enzymes) to synthesize the first strand of the cDNA from the mRNA template.
  • RNA-dependent DNA polymerase enzymes more commonly termed reverse transcriptase enzymes
  • Reverse transcriptase enzymes cannot initiate nucleic acid synthesis de novo. Rather, these enzymes add the first nucleotide of the nascent cDNA strand to the hydroxyl group of a molecule which is bound to the RNA template molecule. This bound molecule to which the enzyme adds the first nucleotide is called a primer and appears to be absolutely required for reverse transcriptase activity - regardless of the source of the enzyme. However, there appear to be major differences in the requirements for priming the initiation of cDNA synthesis under in vitro and in vivo conditions.
  • oligonucleotide primer a short segment of deoxyribonucleic acid (DNA) , termed an oligonucleotide primer, is most commonly used. Frequently this primer is a polymer of deoxyribothymidylic acid (oligo(dT)). In all cDNA protocols currently available this DNA primer is designed simply to anneal to the RNA template molecule, and is not capable of forming a specific structure which contributes to the binding of the reverse transcriptase enzyme to form a primer-enzyme complex (Kimmel and Berger (1987) ; Okayama, H. , et al .
  • in vitro cDNA synthesis may utilize an oligonucleotide primer that is complementary to other sequences within the RNA molecule; however, because of the extensive stretch of complementary nucleotides necessary for annealing to occur, such a primer will be "sequence specific" for the mRNA molecule to which it is designed to anneal. Synthesis of such a sequence specific primer requires prior knowledge of the nucleotide sequence of part of the mRNA. The primer requirements of reverse transcriptase enzymes are discussed in Chapter 5 of Sambrook, et al . (1989).
  • the reverse transcriptase enzymes are already complexed with a structure-specific cellular tRNA molecule which the virus scavenged from a host cell during the previous infection.
  • Transfer RNA molecules are short (70 to 80 nucleotides long) RNA molecules which are folded into complex three-dimensional structures; their usual cellular role is in the translation process (Darnell et al . (1989), chapter 4).
  • This reverse transcriptase enzyme complex is, in turn, bound to the site of cDNA synthesis initiation on the viral RNA template molecule, and serves as the primer-reverse transcription enzyme complex.
  • the reverse transcriptase-tRNA complex acts as primer for an in vivo (in the cell) reverse transcriptase reaction, using the viral RNA molecule, to which this complex is already bound, as the RNA template.
  • the initial cDNA product made from this in vivo reaction is a short cDNA segment termed the "minus-strand strong-stop".
  • This cDNA then serves as a primer molecule in an intramolecular or intermolecular priming event which allows resumption and completion of cDNA synthesis on the retroviral RNA template.
  • the cDNA molecule is then converted to double-stranded DNA by the same reverse transcriptase enzyme complex, and integrated into a chromosome of the infected cell.
  • the initial product of the in vivo cDNA synthesis reaction in hepadnaviral replication is the formation of a "minus- strand DNA" product, and, again, similar to the strategy employed by retroviruses , this initial cDNA product acts to prime at a second polynucleotide site to recommence cDNA synthesis.
  • VL30 virus-like 3OS
  • the hepadnaviruses reverse transcriptase enzymes appear to require specific structural target elements within the hepadnaviral RNA template molecule in order to bind and initiate cDNA synthesis (Wang and Seeger, J. Virol . 67:6507-6512 (1993)).
  • the product of the initial reverse transcriptase reaction in vitro is a single-stranded complementary DNA copy of the mRNA molecule. This reaction is often referred to as "first strand cDNA synthesis.” Thereafter, various techniques are used to generate the second strand of the cDNA.
  • dsDNA double-stranded DNA
  • vector a "vector” which allows growth, selection, and amplification of each copy.
  • Most commonly used techniques eg. Okayama and Berg, Molecular and Cellular Biology 2:161-170 (1982)
  • the mRNA template is eliminated to allow synthesis of the second strand of DNA and thereby form a double-stranded cDNA molecule; specific DNA linkers are then attached to the blunted end of the double-stranded cDNA,and the cDNA is ligated into a suitable cloning vector.
  • the reverse transcriptase-catalyzed step of making a cDNA copy of the mRNA is always performed under in vitro conditions.
  • the quality of the cDNA synthesis depends upon the fidelity and the processivity of the enzyme chosen, and the conditions under which the reaction is performed.
  • Clearly less than full-length cDNA is not acceptable, and a high error rate will compromise the utility of the cDNA produced.
  • Reverse transcriptase enzymes unlike their cellular DNA polymerase counterparts, lack enzymatic 3' -» 5' exonuclease (proofreading) activity so fidelity depends on base discrimination during polymerization.
  • the in vitro fidelity of MuLV reverse transcriptase has been estimated to be 10 "4 (i.e. one wrong nucleotide per 10,000 bases or 10 errors per lOOkb) , and recent studies have determined that the in vivo fidelity is approximately 2xl0 "5 (1 error for every 50,000 bases copied, 2 errors per lOOkb; Mont et al., J. Virol . 66:3683-3689 (1992)).
  • none of the presently known techniques for cDNA synthesis facilitate the priming and initiation of cDNA synthesis by providing a structure- and reverse transcriptase-specific polynucleotide target molecule.
  • the present techniques for cDNA synthesis provide a mechanism whereby inclusion of genetic elements is possible using a polynucleotide template primer molecule whose cDNA product acts as a primer molecule at a second polynucleotide site where cDNA synthesis recommences and completes the linkage of the complementary DNA of the initial, and any subsequent polynucleotide molecules.
  • present techniques for cDNA synthesis are limited by either the requirement that the mRNA be extracted, purified, or withdrawn from cells, or the performance of the reverse transcriptase enzyme under in vitro conditions. These factors limit: the ease of cDNA synthesis; the efficiency of cDNA synthesis (therefore requiring a larger number of cells to construct a representative cDNA library) ; the size of cDNA molecules that can be produced (thereby the genes that are readily clonable by this technique) ; the accuracy of cDNA synthesis in determining which genes are expressed under particular conditions; and the fidelity of the cDNA produced.
  • the present invention relates to methods and compositions for the synthesis of complementary DNA copies of polynucleotide templates.
  • a method for synthesizing a complementary DNA copy of a plurality of polynucleotide molecules is provided.
  • the method comprises providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, the first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to the first region which enables a cDNA product of said molecule to anneal to at least one distinct polynucleotide template molecule.
  • at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template primer molecule is provided.
  • a mixture comprising the reverse transcriptase enzyme or enzyme complex and the polynucleotide template molecules is then formed and incubated under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to the polynucleotide template molecules.
  • a further aspect of the invention provides a method for producing a complementary DNA copy of a polynucleotide template molecule.
  • the method comprises providing at least one polynucleotide template molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, the first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to the first region which enables a cDNA product of the molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to the first region, which third region is sufficiently similar in sequence to the second region such that the complementary DNA product of the second region anneals to the third region so as to allow complementary DNA synthesis to recommence.
  • At least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template primer molecule is provided.
  • a mixture comprising the reverse transcriptase enzyme or enzyme complex and the polynucleotide template molecule is then formed and incubated under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to the polynucleotide template molecule.
  • This method comprises providing a reverse transcriptase-cognate primer transfer RNA molecule in which the 3'-region has been modified to selectively anneal to a subpopulation of cellular polynucleotide template molecules.
  • at least one reverse transcriptase enzyme which initiates DNA synthesis from the primer molecule is provided.
  • a mixture comprising the reverse transcriptase enzyme and the primer molecule is then formed in a viable cell and incubated under conditions which permit the synthesis of DNA molecules which comprises regions complementary to the cellular polynucleotide template molecules. Thereafter, the cDNA products thus produced are analyzed to form a pattern therefrom.
  • a method is accomplished by providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a subpopulation of cellular polynucleotide template molecules.
  • This primer molecule is then utilized in place of the reverse transcriptase- cognate primer transfer RNA molecule in the method just described.
  • compositions useful in the practice of the present method include polynucleotide template primer molecules and recombinant viral particles which are capable of initiating in vivo cDNA synthesis upon introduction into a viable eukaryotic cell.
  • recombinant viral particles comprise at least one polynucleotide template primer molecule which includes the functional sequence equivalents of the sequence elements required for viral packaging (e.g.
  • recombinant viral particles will find use in a mixture which provides all other required trans-acting elements (e.g. capsid, nucleocapsid proteins) which will result in the packaging of the polynucleotide template primer molecule into an infectious particle.
  • compositions provided in the invention include Genetic Elements (GE) template primer polynucleotide molecules which bind at least one reverse transcriptase enzyme or complex thereof and serve as a template molecule for the synthesis of a complementary DNA copy of the template primer molecule.
  • the cDNA product of the 5'-region of this template primer molecule will then act to prime the resumption of cDNA synthesis at least one other site on a polynucleotide molecule.
  • the cDNA synthesis reaction covalently links the cDNA products of at least one polynucleotide template.
  • Compositions of the invention also include DNA molecules and recombinant DNA vectors encoding the polynucleotide molecules of the invention, and kits containing such compositions.
  • Figure 1 depicts a simplified diagram of the eukaryotic RNA transcription process, illustrating removal of the non-coding intron sequences during mRNA processing;
  • FIG. 2 depicts the cloning of gene cassettes in the present invention, in which
  • Figure 2A depicts a schematic diagram of certain techniques involved in cloning of the tRNA Pro gene cassettes.
  • Figure 2B depicts the structure and sequence of a tRNA molecule corresponding to the "wild type" murine tRNAlJTM utilized by Moloney murine leukemia virus, in which the solid line defines the 3' nucleotide sequence which anneals to the virion RNA template and primes the retroviral reverse transcriptase reaction;
  • Figure 3 depicts the relationships involved in reverse transcriptase, tRNA primer, and RNA template interactions necessary for cDNA synthesis, in which Figure 3A depicts retroviral cDNA synthesis sequences for both the 3'-region of the cognate tRNA
  • Figure 3B depicts in vivo cDNA synthesis from a polyadenylated messenger RNA, in which sequences are shown for both the 3'-region of the which has been modified and synthesized in vitro, and the mRNA polyadenylated 3'-segment to which the tRNA binds, as well as the initial stages of in vivo cDNA synthesis (the dashed line) ;
  • Figure 4 depicts the steps involved in the amplification-mutagenesis of the tRNA-encoding promoter- tRNA cassette, wherein the first and second primers are represented by dashed lines with arrows delineating the 5' to 3' orientation, the second primer is represented by a bent dashed line to illustrate the inability of 5' bases to anneal to uncomplimentary bases in the initial template DNA molecule, and the bottom schematic represents the amplified and modified transcription cassette in which the 3'-most bases have been changed to the prescribed sequence encoded in the second primer, and the 3'-end of
  • Figure 5 is a photographic reproduction of an ethidium bromide-stained 1% agarose/TAE gel of in vivo cDNA products from insect Sf9 cells, in which lanes 1 and 10 represent a lkb ladder (BRL) , lanes 2 and 6 represent control ([ ⁇ - 32 P]dCTP alone), lanes 3 and 7 represent control ([ ⁇ - 3 P]dCTP + reverse transcriptase), lanes 4 and 8 represent control ([ ⁇ - 3 P]dCTP + reverse transcriptase + tRNA Mt ) , lanes 5, 9, 11 and 12 represent experimental with modified tRNA primer ([c.- 32 P]dCTP + reverse transcriptase + tRNA p ⁇ ) .
  • lanes 1 and 10 represent a lkb ladder (BRL)
  • lanes 2 and 6 represent control ([ ⁇ - 32 P]dCTP alone)
  • lanes 3 and 7 represent control ([ ⁇ - 3 P]dCTP + reverse transcriptase)
  • the samples in lanes 11 and 12 are not treated with RNase A, the samples in lanes 2- 5, and 11 are electroporations in 0.4cm cuvettes, and the samples in lanes 6-9, and 12 are electroporations in 0.2cm cuvettes;
  • Figure 6 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 5;
  • Figure 7 is a photographic reproduction of an ethidium bromide stained 1% agarose/TAE gel of in vivo cDNA products from Hamster (CHO) cells, in which lanes 1 and 10 represent a lkb ladder (BRL) , lanes 2 and 6 represent control ([ ⁇ _3 P]dCTP alone), lanes 3 and 7 represent control ([ ⁇ - 3 P]dCTP + reverse transcriptase).
  • lanes 1 and 10 represent a lkb ladder (BRL)
  • lanes 2 and 6 represent control ([ ⁇ _3 P]dCTP alone)
  • lanes 3 and 7 represent control ([ ⁇ - 3 P]dCTP + reverse transcriptase).
  • lanes 4 and 8 represent control ([ ⁇ _32 P]dCTP + reverse transcriptase + tRNA yt )
  • lanes 5 and 9 represent experimental with modified tRNA primer ([ ⁇ 32 P]dCTP + reverse transcriptase + tRNA pol ⁇ )
  • Figure 8 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 6;
  • Figure 9 is a photographic reproduction of an ethidium bromide stained 1% agarose/TAE gel of in vivo cDNA products from Hamster (CHO) cells, in which lanes 1 and 8 represent a lkb ladder (BRL) , lanes 2 and 3 represent control ([ ⁇ -32 P]dCTP + reverse transcriptase + tRNA ut ) , lanes 4 and 5 represent experimental with modified tRNA primer ([ ⁇ _3 P]dCTP + reverse transcriptase + tRNA polyU ) , and lanes 6 and 7 represent control ([ ⁇ " 32 P]dCTP + reverse transcriptase + oligo(dT) (5//g) .
  • the samples in lanes 3, 5, and 7 are treated with SI nuclease;
  • Figure 10 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 9;
  • Figure 11 depicts a simplified diagram of retroviral proviral synthesis.
  • the dashed line represents the viral RNA genomic segment with the tRNA primer (looped structure) and reverse transcriptase (RT) annealed to the complementary sequence of the retrovirus-specific Primer Binding Site (PBS) .
  • the "R" regions are repeated sequences present near each end of the genomic RNA segment.
  • the heavy solid lines represent cDNA synthesis directed by the RNA template sequences;
  • Figure 12 depicts a simplified diagram of a representative embodiment of in vivo cDNA synthesis in the presence of Vector Control Elements (VCE) RNA template.
  • VCE Vector Control Elements
  • the dashed line represents an RNA molecule.
  • PolyA tract refers to polyadenylic acid — one choice for the 5'-terminal sequence of the RNA template molecule.
  • Neo refers to an RNA sequence which codes for a region which confers biological resistance to the antibiotic G418 in eukaryotes (neomycin in prokaryotes) .
  • Ori is a commonly accepted abbreviation for the origin of replication of a polynucleotide molecule in a biological host.
  • the arrow which encompasses the word "promoter” indicates one orientation of a promoter element operatively linked to any polynucleotide segment which encodes a biological molecule; and
  • Figure 13 depicts aspects involved in the generation of a primer-specific cell expression pattern in viable cells, in which ,
  • Figure 13A provides a diagrammatic representation of
  • RNA transcripts and their associated sizes present in cell populations A and B; and Figure 13B depicts one possible pattern of cDNA products resulting from the in vivo cDNA synthesis employing a specific template primer or modified cognate tRNA primer molecule.
  • the present invention relates to methods and compositions for the synthesis of complementary DNA (cDNA) .
  • This invention provides, for the first time, a method by which cDNA synthesis is made possible utilizing a structure-specific analog to the natural target site of the reverse transcriptase enzyme or reverse transcriptase enzyme complex.
  • suitable template, or template primer molecules which have a first site which binds to a reverse transcriptase enzyme or reverse transcriptase enzyme complex is introduced into a mixture in the presence of a reverse transcriptase enzyme, or reverse transcriptase enzyme complex, and the mixture is incubated under conditions such that a DNA molecule complementary to at least one polynucleotide template molecule(s) is produced.
  • the requirements for priming cDNA synthesis in an in vivo environment appear to be more stringent than in vitro, and oligo(dT) primer molecules, and other DNA primers appear not to function under in vivo cDNA synthesis conditions (see Example section) .
  • all known reverse transcriptase enzymes functioning under in vivo conditions are directed to the site of cDNA synthesis initiation by a structure- specific polynucleotide molecule.
  • the first step involved in initiation of all in vivo cDNA synthesis reactions appears to be the directed binding of the reverse transcriptase enzyme to a structure-specific polynucleotide target.
  • this role of structure-specific polynucleotide molecule is satisfied by cellular molecule(s) referred to as the reverse transcriptase cognate transfer RNA (tRNA) molecule(s) .
  • cognate tRNA molecules they are designated "cognate” for two reasons: (1) the reverse transcriptase enzyme produced by that specific retrovirus recognizes and binds to the specific structure of a very limited subset of cellular tRNA molecules, and (2) the 3'-region of the cognate tRNA molecule, in turn, binds to a complementary sequence at the site of cDNA initiation (Primer Binding Site) which is specific for the retrovirus, and is encoded in the retrovirus RNA genomic template molecule.
  • cDNA initiation Primary Binding Site
  • hepadnavirus pregenomic RNA template specific binding sites epsilon (e)
  • epsilon e
  • the polynucleotide template, or template primer molecule is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • the polynucleotide template or template primer molecule is a modified retroviral or hepadnavirus template RNA molecule.
  • the invention provides, for the first time, a means by which all of the genetic elements necessary for biological selection, replication and amplification, integration and expression of an heterologous genetic element can be covalently included as a contiguous cDNA product in an in vivo process; therefore many of the crucial steps of molecular cloning are performed by the viable cell in the in vivo intracellular environment.
  • This genetic control information is encoded in a Genetic Elements (GE) polynucleotide template, or template primer molecule.
  • GE Genetic Elements
  • this polynucleotide template, or template primer molecule is transcribed within the viable cell.
  • this GE polynucleotide template, or template primer molecule is introduced into a viable target cell in the presence of a suitable reverse transcriptase enzyme, or reverse transcriptase enzyme complex.
  • the GE polynucleotide template, or template primer molecules when used in combination with a retroviral reverse transcriptase enzyme and a modified cognate tRNA molecule, may include sequence elements which will form structures similar to those of the U5-leader and U5-IR stems in the retroviral genomic RNA template. These sequences have been shown to be important in viral replication (Cobrink, D. , et al . , J. Virol . 62:3622-3630 (1988)), and appear to interact with the T ⁇ C loop of the tRNA primer to significantly improve the efficiency in priming cDNA synthesis (Cobrink, D. , et al . , J. Virol .
  • a modified tRNA primer molecule may interact in an energetically more favorable manner with RNA template sequences which reproduce an analogous RNA structure.
  • inclusion of nucleocapsid protein (or other proteins) may facilitate the formation of an initiation complex composed of the RNA template, tRNA primer and reverse transcriptase enzyme (Meric and Goff, J. Virol . 63:1558-1568 (1989); Khan and Giedroc, J. Biol . Chem. 267:6689-6695 (1992)), thus improving the efficiency of cDNA synthesis.
  • the GE polynucleotide template, or template primer molecules when used in combination with a hepadnavirus reverse transcriptase enzyme they may include sequence elements which will form structures similar to those of the specific binding site, epsilon (e) pregenomic RNA template. These sequence elements have been shown to be critical for both the initiation of cDNA synthesis, and packaging of the hepadnavirus genome. Inclusion of sequences in the 5'-end of the initial polynucleotide template molecule which are represented in the 3'-end of the same template molecule will facilitate intramolecular annealing of the initial minus-strand strong-stop cDNA product with these 3'-sequences. This will result in intramolecular priming at a second polynucleotide site, where resumption of cDNA synthesis will occur.
  • epsilon (e) pregenomic RNA template These sequence elements have been shown to be critical for both the initiation of cDNA synthesis,
  • template indicates a nucleotide sequence from which a complementary sequence is produced.
  • template primer indicates a polynucleotide sequence from which cDNA synthesis is initiated and extended, and the cDNA product of said template primer molecule serves to prime the resumption of cDNA synthesis at a second polynucleotide site.
  • Genetic Elements (GE) and Vector Control Elements (VCE) template primer molecule are used interchangeably and indicate a template primer molecule which may include encoded genetic regions for functions including, but not limited to: amplification, selection, replication, insertion, segregation, integration, excision, stabilization, purification, expression, detection, localization, processing, or packaging in prokaryotic or eukaryotic cells.
  • analog is used to indicate any sequence- specific representative of a naturally-occurring nucleotide.
  • reverse transcriptase enzyme is taken to mean any polymerase which can catalyze the addition of a deoxynucleotide or analog thereof to a primer annealed to an RNA template.
  • polynucleotide includes homo- and hetero- polymers of deoxyribonucleic acids, ribonucleic acids and analogs thereof.
  • modified reverse transcriptase-cognate primer transfer RNA molecule refers to any tRNA molecule, whether produced chemically, by means of an artificial biological system, or purified from a biological source, which is modified so as to prime the activity of a specific reverse transcriptase enzyme.
  • Primer Binding Site refers to the specific nucleotide sequence in an RNA template molecule to which the naturally-occurring cognate primer of a specific reverse transcriptase enzyme anneals to initiate cDNA synthesis. Therefore, any nucleotide additions, deletions or other modifications in the naturally- occurring cognate primer which result in variations in the RNA template sequence to which the primer anneals constitute binding at a site other than the Primer Binding Site.
  • R region sequence refers to the natural repeated sequences found at both the 3'- and 5'-ends of retroviral genomic RNA.
  • expression cassette refers to a DNA construct which includes all sequences necessary for the expression of the coded product. Accordingly, an expression cassette will include DNA encoding at least a promoter region, the sequence of interest and a transcription termination region.
  • vector and plasmid are interchangeably used to include any means which permits DNA to be replicated and selected in a particular system.
  • operative linkage refers to nucleotides which are joined in a manner which preserves the functional relationship between the sequences on each side of the linkage.
  • a promoter operatively linked to a DNA sequence will be upstream both with respect to the direction of transcription and with respect to the transcription initiation site and inserted in a manner such that transcription elongation proceeds through the DNA sequence.
  • cDNA synthesis does not require the isolation of mRNA from cells.
  • Such cDNA synthesis eliminates the problems associated with conventional in vitro techniques where: 1) mRNA isolation is required; 2) the cellular components are withdrawn into an artificial environment; or, 3) the cells are killed and subjected to harsh conditions. Therefore, the mRNA templates are more likely to be intact and full length cDNA clones can more reliably be obtained.
  • in vivo cDNA synthesis does not require the in vitro activity of reverse transcriptase, but rather permits the reverse transcription step to be performed in vivo (i.e. within the cellular environment) , such that the efficiency, fidelity and processivity of the reverse transcriptase enzyme is optimized.
  • cDNA synthesis requires isolation and purification of mRNA from cells, or the withdrawal of the reaction components into an artificial environment, followed by an in vitro cDNA synthesis step.
  • the requirement for a primer for the reverse transcriptase enzyme is most commonly met by supplying an oligo(dT) primer molecule that anneals to the poly(A) tail of mRNA molecules, or an oligonucleotide molecule that is complementary to a known portion of target mRNA sequence.
  • oligonucleotide primers appear to be effective in vivo (see Examples below) .
  • Simple oligomer primers do not contain this structural requirement, and so, the retroviral reverse transcriptase enzymes may not recognize and associate with the primer under in vivo conditions, even if the polynucleotide primer is able to anneal to the mRNA template.
  • hepadnaviral reverse transcriptase enzymes recognize and bind to a specific structural region, epsilon (e) , present on the pregenomic RNA template molecule (Weber, M. et al., J. Virol . 68:2994- 2999 (1994)).
  • Each infective retrovirus particle contains two copies of a single-stranded viral RNA chromosome each of which is associated with a specific host tRNA molecule which anneals to a particular region of the retroviral RNA termed the Primer Binding Site.
  • a sequence of bases at the 3'-end of the tRNA anneals to the Primer Binding Site of the retroviral RNA.
  • the reverse transcriptase enzyme (which is already associated with this complex) then utilizes this tRNA as a primer molecule, adding the first nucleotide of the nascent DNA molecule to the 3'-hydroxy terminus of the tRNA.
  • the specificity of priming retroviral reverse transcription is determined by the base pair sequence at the 3'-end of the tRNA molecule which anneals to the retroviral genome.
  • Each retrovirus utilizes a tRNA primer capable of annealing to the specific Primer Binding Site sequence present in the retrovirus genome.
  • the human immunodeficiency virus (HIV) genome utilizes tRNA ⁇ as a primer.
  • the eighteen nucleotides at the 3'-terminal of the tRNA ⁇ ys are complementary to the HIV Primer Binding Site sequence.
  • portion(s) of the same tRNA molecule will be recognized by the viral reverse transcriptase enzyme so that a trimolecular complex is ultimately formed (tRNA primer-reverse transcriptase-RNA template) .
  • a polynucleotide template or template primer molecule should fulfill two criteria. Firstly, the molecule should be able to bind a reverse transcriptase enzyme or reverse transcriptase enzyme complex; secondly, the bound enzyme or enzyme complex must initiate cDNA synthesis from the region of that site and produce an initial cDNA molecule which is complementary to the polynucleotide template.
  • the simple oligonucleotide primers presently used with retroviral reverse transcriptase enzymes to initiate in vitro cDNA synthesis do not function in vivo (see data, included) .
  • tRNA molecules are able to function as primers for the in vivo action of reverse transcriptase on the retroviral genome
  • these tRNA primers anneal specifically to the retroviral Primer Binding Site and are not designed to anneal to sequences present in all mature cellular mRNA molecules.
  • reverse transcriptase enzymes from hepadnaviruses will recognize and bind to the specific binding site (e) present in the hepadnaviral pregenomic RNA template molecule, but will not bind or initiate cDNA synthesis from regions present on all mature cellular mRNA molecules.
  • the present invention provides a method of in vivo cDNA synthesis by providing template primer molecules whose cDNA products will anneal to all polyadenylated cellular mRNA molecules, and are utilized by the reverse transcriptase enzyme such that a cDNA copy of the mRNA molecule to which the primer molecule anneals is produced.
  • This invention is analogous to retroviral and hepadnaviruses replication processes (actually in vivo cDNA synthesis reactions) substituting modified retroviral or hepadnaviral template molecules, or template primer molecules, for the viral template molecules.
  • the template primer molecule is a modified retroviral template molecule wherein the 5'-region of the molecule is replaced with a polyadenylic acid sequence such that the cDNA product of the template anneals to the 3'- poly(A) tail that is present on mature cellular mRNA molecules.
  • the template primer molecule is a modified hepadnaviral template molecule wherein the 5'-region of the molecule is replaced with a polyadenylic acid sequence such that the cDNA product of the template anneals to the 3'- poly(A) tail that is present on mature cellular mRNA molecules.
  • Such preferred template primer molecules may be used to synthesize in vivo cDNA copies of all mature mRNA molecules contained within a viable cell. Because the product of this template primer will anneal to all mature mRNA molecules, the template primer may be used to produce a cDNA library, that is a collection of cDNA molecules that represents all of the structural genes being expressed in the cell at that given point in time. In one embodiment of the invention the cDNA synthesis reaction takes place in viable cells under in vivo conditions, without subjecting the cells to the stress and perturbation of mRNA extraction, isolation, or withdrawing the cellular contents into an artificial in vitro environment.
  • the cDNA synthesis products more accurately reflect the snapshot of mRNA populations within the cell at the time that the cDNA synthesis was initiated, and the products are synthesized in an in vivo environment.
  • an initial specific polynucleotide template primer molecule is utilized to both increase the annealing and priming potential of the nascent cDNA transcript, and to provide a means to incorporate additional encoded genetic elements, or vector control elements into the final in vivo cDNA products.
  • the 5'-region of the template primer molecule is modified so that it is complementary to part of a specific polynucleotide sequence.
  • Synthesis of specific cDNA molecules may be useful in many different ways, including, but not limited to the following:
  • a sequence-specific template primer may be utilized to clone cDNAs with known sequence, to clone from genes which represent a specific gene family (all of which share a common sequence) , or to determine ratios of splice variants on two separate unrelated transcripts.
  • a template primer molecule can be used for intracellular mutagenesis or cDNA coupling reactions; a template primer molecule which encodes desired sequence elements 3' to a 5' sequence, the cDNA product of which will anneal to a distinct cellular transcript and prime the resumption of cDNA synthesis 5' to a region which one desires to mutate, will allow replacement of the cellular transcript sequences 3' to the site of template primer cDNA product annealing - with the concomitant loss of the cellular transcript information 3' to the site of this template primer annealing.
  • a template primer having a sequence which includes a number of polyadenylic acid residues located 3' to a 5'- terminal specific polynucleotide sequence will allow the cDNA product of the template primer molecule to reposition itself and act as a primer on a second polynucleotide template molecule (e.g. at the junctional region proximal to the polyadenylic acid stretch present on mature messenger RNAs) , such that a snapshot of members containing a complementary junctional sequence will be converted to cDNA product.
  • RNA template size and frequency RNA template size and frequency
  • sensitivity of cDNA product detection choice of nucleotide analog incorporated
  • priming at regions in the mRNA template other than at the 3'-end priming at regions in the mRNA template other than at the 3'-end
  • other factors affecting the efficiency of cDNA synthesis are examples.
  • this technology allows a primer-specific cell expression pattern to be developed.
  • the pattern will be specific for the type or developmental stage of a cell or tissue.
  • the pattern may be diagnostic for cellular perturbation (e.g. transformation or infection) , or change in response to an applied stimulus. Changes may be characterized solely by changes in the density of various cDNA product which make up the pattern (See Figure 13) .
  • RNA template primer which is selective for an RNA template can be used for in vivo DNA sequencing reactions to determine the 5'-sequence of a desired RNA template, without the need to clone and isolate the template.
  • PCR polymerase chain reaction
  • a specific primer can then be used to initiate in vivo cDNA synthesis within the aberrant cell as a form of antisense therapy based upon the ability of the reaction to convert existing mRNA templates into a cDNA product which is no longer capable of being translated.
  • an HIV-specific complementary primer could incorporate modified deoxynucleotide bases into a cDNA which could be used for detection, sequencing or sorting (Link, H, et al . , J. Med. Virol . 37:143-148 (1992); Prober, J.M. et al .
  • the present invention includes template and template primer molecules that may be used for in vivo cDNA synthesis. More specifically and in preferred embodiments, these molecules are modified retroviral or hepadnaviral RNA template molecules.
  • any polynucleotide that is capable of (a) binding a reverse transcriptase enzyme or enzyme complex and (b) acting as a template for a reverse transcriptase enzyme or enzyme complex such that initiation and synthesis of a DNA molecule complementary to that polynucleotide template molecule can occur, can be utilized in the cDNA synthesis method of the present invention.
  • the suitability of a particular template or template primer molecule for use in cDNA synthesis may be assessed by performing cDNA synthesis as described below and assaying the products of that synthesis.
  • the template (or template primer) and the reverse transcriptase enzyme (or reverse transcriptase enzyme complex) may either be present in the cell, or either one or both can be synthesized in or introduced into the cell.
  • in vivo cDNA synthesis may be performed in a cell line that carries genes encoding a reverse transcriptase enzyme and the template (or template primer) molecule. The expression of these genes will result in the synthesis of the reverse transcriptase enzyme and the template (or template primer) in the cell.
  • the genes encoding the reverse transcriptase and the template (or template primer) molecules may be expressed under the control of inducible promoters such that the induction of the genes will lead to the expression of the reverse transcriptase and the template molecules and thereby initiate cDNA synthesis.
  • the template (or template primer) molecules and the reverse transcriptase enzyme (or reverse transcriptase enzyme complex) will be introduced into the cell from an external source.
  • a number of techniques have been established for the delivery of biological materials into viable cells. Many of these techniques have been described (Molecular Cloning; A Laboratory Manual Cold Spring Harbor Laboratory Press,
  • techniques for delivery of the in vivo cDNA synthesis primers, templates and template primers into cells includes electroporation and liposomal transfection. These technique can be used, as described below, to introduce modified tRNA primers, reverse transcriptase enzymes, and modified deoxynucleoside triphosphates into a number of distinct cell types.
  • the reverse transcriptase enzyme is introduced into the cell from an external source, the choice of in vivo cDNA synthesis primers, templates and template primers will be determined by the type of the reverse transcriptase enzyme selected.
  • Retroviral reverse transcriptase Avian Myeloblastosis Virus reverse transcriptase and Moloney Murine Leukemia Virus reverse transcriptase. These enzymes are available on a commercial basis from such vendors as: Stratagene, 11011 N. Torrey Pines Road, La Jolla, California 92037; Bethesda Research Laboratories, Inc., P.O. Box 6009, Gaithersberg, Maryland, 20877; New England Bio Labs, Inc., 32 Tozer Road, Beverly, Massachusetts, 01915; and Boehringer Mannheim Biochemicals, 9115 Hague Road, P.O. Box 50816, Indianapolis, Indiana, 46250.
  • the various enzymes have both inherent parameters: fidelity (error rate) , processivity (ability to complete cDNA synthesis) , and structure (heterodimer vs monomer) ; and marketing parameters, such as availability and acceptance, which are factors which should be considered in choosing the enzyme.
  • fidelity error rate
  • processivity ability to complete cDNA synthesis
  • structure heterodimer vs monomer
  • marketing parameters such as availability and acceptance, which are factors which should be considered in choosing the enzyme.
  • it may be important to use a reverse transcriptase enzyme derived from a retrovirus or retroelement which functions in the cell type and organism of the embodiment e.g. use a reverse transcriptase enzyme from a human retrovirus to synthesize cDNA in human cells.
  • a DNA cassette containing the encoded modified tRNA sequence operatively linked to a promoter sequence was cloned into a bacterial vector. This allowed production of the modified tRNA primer in vitro, using an RNA polymerase which recognized the promoter sequence.
  • the choice of promoter for use in instances where the in vivo cDNA synthesis primer is to be transcribed in vitro will be dictated by the polymerase enzyme to be used in the selected in vitro transcription system.
  • the modified tRNA pro GGG primer was expressed under the control of a bacteriophage T7 promoter in an in vitro transcription system using the T7 RNA polymerase enzyme.
  • the modified tRNA primer produced was isolated and introduced into cells along with the reverse transcriptase enzyme, a modified MoMLV template primer molecule and radiolabeled deoxynucleotide. Following introduction of these exogenous components, the cell was incubated for a sufficient period of time and under suitable conditions to allow in vivo cDNA synthesis to occur. Suitable conditions are generally those under which the cell type in question is usually grown. In preferred embodiments of the present invention, the target cell is incubated for 0.5-2 hours under normal culture conditions for that cell type following introduction of the primer and reverse transcriptase enzyme.
  • the introduction of a radiolabeled dNTP may be used to determine the efficacy of in vivo cDNA synthesis under particular conditions and with particular primers.
  • a radiolabeled dNTP for example, [ ⁇ - 32 P]dCTP
  • the cDNA produced in vivo was then extracted from the target cell utilizing the Hirt- extraction protocol (Hirt, B. , J. Mol . Biol . 26:365-369 (1967)).
  • Hirt- extraction protocol Hirt, B. , J. Mol . Biol . 26:365-369 (1967)
  • the extracted cDNA product appeared to be double-stranded nucleic acid which was successfully cloned using common cloning procedures.
  • the primer, templates and template primers and/or enzyme could be produced from genes introduced into cells; the enzyme could be expressed from a gene within the cell, and the primer, templates and template primers introduced into the cell; or the primer, templates and template primers could be expressed within the cell and the reverse transcriptase could be introduced from the outside.
  • modified deoxynucleoside triphosphates were used as a tool to follow the incorporation of deoxynucleotides and is not a requirement of the method; however, it should be noted that inclusion of modified deoxynucleotides provides both a convenient technique of following incorporation and synthesis (when ⁇ -labeled deoxynucleotides are used) , and provides a means of selecting or distinguishing the products of an in vivo cDNA incorporation reaction (using biotinylated or other deoxynucleotide analogs) .
  • the products of these reactions can be used in various ways including, but not limited to: the construction of subtractive cDNA libraries; production of specific cytotoxic or light sensitive cDNA products; in vivo DNA sequencing; and cDNA probes for analytical, diagnostic or preparative use.
  • M (Molar) ; mM (millimolar) ; ⁇ V (micromolar) ; N (Normal) ; mol (moles) ; mmol (millimoles) ; //mol (micromoles) ; nmol (nanomoles) ; kg (kilograms) ; gm (grams) ; mg (milligrams) ; ⁇ g (micrograms) ; ng (nanogra s) ;
  • L (liters) ; ml (milliliters) ; ⁇ l (microliters) ; cm (centimeters) ; mm (millimeters) ; jtm (micrometers) ; nm (nanometers) ; V (volts) ; ⁇ F (microfarads) and °C (degrees Centigrade) .
  • restriction enzymes T4 DNA ligase, and polynucleotide kinase are obtained from New England
  • the [ ⁇ 32 P]dCTP, [ ⁇ 32 P]UTP, and [ ⁇ - 32 P]ATP are obtained from Amersham Corporation, and Moloney Murine Leukemia Virus reverse transcriptase buffers, liposome preparations and enzymes are obtained from Bethesda Research Laboratories. These materials are used according to manufacturer's instructions unless otherwise specified. Electroporation is performed using an electroporator from Invitrogen Corporation, and electroporation cuvettes from BioRad Corporation. Oligonucleotides are obtained from Operon Technologies, Inc, or can be synthesized, e.g., on an Applied Biosystems DNA synthesizer according to the manufacturer's instructions. Thermus aquaticus DNA polymerase I is obtained from Perkin-Elmer Cetus. All standard molecular biology techniques are performed according to Sambrook et al . (1989) or Berger and Kimmel (1987), herein incorporated by reference.
  • Nucleic acid sequences disclosed herein are divided into 10-mer or smaller oligonucleotides as a matter of convenience, and should be interpreted as continuous sequences unless otherwise indicated.
  • EXAMPLE 1 I. Synthesis of genes encoding tRNA primers
  • the Moloney Murine Leukemia virus utilizes the murine tRNA ⁇ TM isoacceptor molecules as primers for synthesis of retroviral DNA (Harada, F. , et al . , J. Biol . Chem. 254:10979-10985 (1979); Peters and Dahlberg, J. Virol . 31:398 (1979)).
  • These particular tRNA molecules are found complexed with the MoMuLV reverse transcriptase enzyme in the virion, and this complex, in turn, anneals to the primer binding site on the viral RNA genome (see Figures 2B and 3A) .
  • the MoMuLV reverse transcriptase capable of utilizing the annealed tRNA molecule as a primer, initiates DNA synthesis, and proceeds to synthesize a DNA molecule that is complementary to the template RNA molecule to which the tRNA primer is annealed.
  • the nucleotide sequence of murine tRNA pro GGG primer is known (Harada et al . , (1979)). Two versions of this tRNA molecule are transcribed in vitro from genetic cassettes which are synthesized using oligonucleotides, then cloned and confirmed by sequence analysis. The deoxyribonucleotide primers used these reactions are depicted below:
  • Primer No. 4 (SEQ ID No. 4): 5'-TGGAATTCTC TTCAAAAAAAAAAA AAAAGAACCC GGGACCTCTC GCACCCCAAG CGAG
  • Primer No. 6 (SEQ ID No. 6) : . 5'-CGAAGCTTTA AAAAAAAAAA AAAAAAAAAG AACCCGGGAC CTCTCGCACC CCAAGCGAG
  • Oligonucleotide primers No. 1 and No. 2 are designed to produce a recombinant tRNA molecule corresponding to the "wild type" murine tRNA pro GGG primer as illustrated in Figure 2A.
  • the in vitro transcribed tRNAs will not contain the modified ribonucleotide bases that exist in their cellular counterparts.
  • these modifications have been found to have the same efficacy in terms of both reverse transcriptase-tRNA interaction, and their ability to prime in vitro cDNA synthesis, as the naturally occurring cognate tRNAs (Barat, C. et al . (1989); Weiss, S. et al . (1992); Barat, C. et al .
  • the 3'-ends of these primers are complementary such that the 3'-ends of primer No. 1 and primer No. 2 can be annealed in vitro, then treated with the Klenow fragment of DNA polymerase I in the presence of dNTPs to produce a double-stranded DNA molecule comprising a T7 RNA polymerase promoter-tRNA wt encoding cassette (see Figure 2A) .
  • This primer pair is designed such that this bacteriophage T7 promoter sequence is operatively linked to the 5'-end of the tRNA molecule.
  • This promoter-tRNA cassette is flanked with restriction sites to allow the cassette to be cleaved from a cloning vector, and an .Earl restriction site is incorporated into the 3'-end of the cassette so that digestion of the cassette with the Earl endonuclease prior to in vitro transcription reactions results in linearization of the template (hence termination of transcription) at the exact 3'-end of the encoded tRNA sequence.
  • the nucleotide sequences of primer Nos. 1 and 2 are set forth in SEQ I.D. Nos. 1 and 2, respectively.
  • a second primer pair (primer Nos. 3 and 4) is designed to encode a modified form of the tRNA molecule, the modified tRNA molecule being termed tRNA polylJ , as follows: SEQ. ID No. 7
  • a bacteriophage T7 promoter sequence is operatively linked to the 5'-end of the tRNA sequence and flanking restriction sites and an .Earl site is incorporated.
  • Primer No. 4 encompassing the 3'-end of the tRNA pol ⁇ j molecule, encodes a poly(U) sequence in place of the 3'-terminal nucleotides of the wild type tRNA molecule (compare the tRNA sequences in Figures 3A and 3B) .
  • Primer Nos. 3 and 4 are set forth in SEQ I.D. Nos. 3 and 4 respectively. Equal molar ratios of primer Nos.
  • kinased oligomer primer pairs Nos. 1 and 2, and primer Nos. 3 and 4 are annealed (80°C for 3 minutes, slowly cooled from 60 ⁇ C to 37 ⁇ C over 20 minutes) in Klenow buffer (50mM Tris-chloride (pH 7.6) at 25 ⁇ C, lOmM MgCl 2 , lOmM >9-mercaptoethanol) , containing 2mM dNTPs.
  • Klenow buffer 50mM Tris-chloride (pH 7.6) at 25 ⁇ C, lOmM MgCl 2 , lOmM >9-mercaptoethanol
  • the annealed primer pairs are then incubated for 30 minutes at 37°C with 10 units of the Klenow fragment of DNA polymerase I in a 30//1 volume to complete double-stranded DNA synthesis.
  • These double-stranded DNA cassettes are then extracted and precipitated using commonly available techniques, resuspended in 1/10 TE buffer (lmM Tris- chloride, O.lmM EDTA (pH 8.0)), and ligated into the dephosphorylated Smal site in the pUCl ⁇ cloning vector (Yannish-Perron et al . , Gene 33:103-119 (1985)).
  • the wild type tRNA gene created by the combination of primer Nos.
  • One technique involves cutting the cloned insert by restriction enzyme digestion. Digestion with Earl restriction endonuclease removes the promoter-tRNA cassette from pUC18-T7tRNA Ht , digesting in vector sequences substantially 5' to the T7 promoter sequence, and digesting precisely at the 3'-end of the tRNA gene template.
  • the above approach may not be used with the pUC18- T7tRNA pol ⁇ j cloning vector.
  • PCR polymerase chain reaction
  • the first primer is a commercially available primer which anneals to sequences in the pUC18 vector located 5' to the inserted T7 promoter region with the 3'-end directed towards the T7 promoter region sequence
  • the second is a "Reverse tRNA Primer" (set forth in SEQ I.D. No. 5) which anneals to bases of the desired T7 promoter-tRNA p ⁇ cassette, such that the 5'-most base of the Reverse tRNA Primer is the last 3'-base of the encoded tRNA pol ⁇ j template, as follows: SEQ. ID No. 8
  • PCR buffer 67mM Tris (pH 9.2 at 25 ⁇ C, 16.6mM (NH 2 S0 4 ,, 1.5mM MgCl 2 ; 50ng of each primer, approximately lxlO 8 molecules of the pUC18-tRNA pol ⁇ j construct, and 250//M concentrations of each of the deoxynucleotide triphosphates.
  • the reaction mixtures are heated to 100°C for 3 minutes, then cooled to 15°C.
  • the tubes are centrifuged briefly to collect the contents, then 1 unit of Taq polymerase and a drop of mineral oil are added to each 20//1 reaction.
  • the reaction is performed in 40 cycles with the following regimen: 1 minute at 94°C; then 1 minute at 55 ⁇ C.
  • the amplified products are pooled from 5 reactions, rendered blunt- ended with Klenow fragment and ImM dNTPs, extracted to remove protein and traces of mineral oil, and EtOH precipitated using standard protocols.
  • the pellet is rinsed with 70% ethanol (EtOH) and dried.
  • the pellet is then resuspended in 1/10 TE, and fragments are examined on a 2% agarose/TAE gel against known size standards.
  • PCR is known to allow incorporation of single nucleotides (usually deoxyriboadenylic acid residues) to the ends of the amplified product in a template-independent manner
  • tRNA Ht For in vitro production of tRNA Ht , reactions are performed using .Earl-linearized pUC18-tRNA Ht template.
  • tRNA pol ⁇ j the PCR-amplified T7-tRNA polyU cassette is used as the template; alternatively, a £>ral sensitive T7-tRNA pol ⁇ , cassette is linearized and used as template for the in vitro transcription reactions (produced with oligomers Nos. 3 and 6, using the same protocols used for the production of the .Earl compatible T7-tRN pol ⁇ j template) .
  • the following conditions may be utilized (Gurevich, V.V. et al . , Anal . Biochem.
  • the tubes are then incubated at 37 ⁇ C for 4 hours.
  • RNase-free DNase is then added to the reactions, and the digestion of template DNA allowed to proceed for 15-30 minutes; at this point, small aliquots of the reaction mixture can be removed in order to determine the efficiency of incorporation. This determination can be achieved by cold trichloroacetic acid precipitation of an aliquot of the reaction mixture in the presence of an excess of RNase-free carrier DNA. The control for this determination, total counts, is performed on unprecipitated material from the same reaction mixture.
  • reaction mixtures 150 l of RNase-free dH 2 0 and 20//1 of 3M NaOAc and the mixtures are extracted with an equal volume of phenol/CHCl 3 (pH 6.5) (phenol buffered at pH 6.5 is used to minimize the possibility of base-catalyzed hydrolysis of the RNA product) , followed by CHC1 3 .
  • the products are precipitated with 2-propanol, the pellets rinsed, and the precipitate dried.
  • the primer product is resuspended in RNase-free dH 2 0, and an aliquot checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards.
  • primers can then be biotinylated, 32 P-labeled, or contain sequence elements such as restriction sites which will be copied and incorporated during second-strand cDNA synthesis following base-catalyzed hydrolysis of the original template (the 2'-0"Methyl primer will protect the annealed complementary portion of the original RNA template as wellj .
  • These modifications do not appear to disrupt the ability of the primers to associate with proteins (Sprout, B.S. et al . , (1989)), and the modified oligoribonucleotides anneal with the expected specificity.
  • the elimination of the 2'-OH group renders an RNA primer more resistant to base-catalyzed (nucleophilic) attack on the neighboring 3',5'- phosphodiester bond, and it is resistant to a variety of ubiquitous RNases.
  • the modified tRNA primer synthesized as described above is used for in vivo cDNA synthesis reactions in numerous different cell lines. Control reactions are also performed in the absence of primer, with an oligo(dT) primer, or with the in vitro-transcribed wild- type tRNA Ht molecule as primer. In these experiments, the primer is introduced into cells via electroporation, along with Moloney Murine Leukemia Virus reverse transcriptase and [ ⁇ - 32 P]dCTP. The radiolabeled dCTP is included to facilitate the analysis of the products of these reactions.
  • the products of the reaction are extracted from the cells by the Hirt extraction technique (Hirt, B. , J. Mol . Biol . 26:365-369 (1967)), and digested to completion with ribonuclease A.
  • Hirt extraction technique Hirt, B. , J. Mol . Biol . 26:365-369 (1967)
  • ribonuclease A ribonuclease A.
  • the product is assessed by quantifying incorporation, and by examining of the size of the products separated on agarose gels, with known size standards, using autoradiography. Further, SI nuclease treatment, and RNase H treatment are used to determine the nature of the DNA product.
  • the cells are suspended at a concentration of lxlO 7 cells/ml in Grace's medium.
  • the reaction components comprising the reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g) and [ ⁇ r 32 P]dCTP (50//Ci) are mixed in a total volume of 50//1 in reverse transcription buffer (obtained from BRL) containing dithiothreitol (DTT) (lOmM) , and incubated at room temperature for 10 minutes.
  • the Sf9 cells (5xl0 6 cells in 0.5ml) are then added to the reaction components and the mixture is transferred into a chilled 0.4cm electroporation cuvette. Electroporation is then performed with the electroporator set at 200V, 250//F and infinite resistance.
  • the radioactive supernatant is carefully removed and disposed of properly, and 1ml of 0.6% sodium dodecyl sulfate (SDS) , lOmM EDTA (pH 7.5) is added to the cell pellet. Immediately this pellet is gently resuspended with a large bore Pipetman P1000 tip (a portion of the tip removed to increase the bore diameter, thus decreasing the shear forces and fragmentation of genomic DNA) . The viscous lysate is placed on ice for 5 minutes, then 250vl of 5M NaCl is added and the tube inverted several times to mix. The tubes are then placed on ice.
  • SDS sodium dodecyl sulfate
  • lOmM EDTA pH 7.5
  • the extract is centrifuged for 20 minutes at top speed in a cold Eppendorf microfuge. The supernatant is carefully withdrawn from the pellet (which is discarded in radioactive waste) , and the supernatant split between two new 1.5ml microfuge tubes. These supernatants are extracted twice with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and 1/6 volume of 2-propanol; the pellets are rinsed with 70% ethanol, and then dried.
  • the pellets are resuspended in 18//1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0) ; the resuspended nucleic acid fraction is treated with ribonuclease A (2/1 of lOmg/ml stock solution), at 37°C for 15 minutes, followed by the addition of 30/1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0). The solution is then extracted once with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and two volumes of ethanol; the pellets are rinsed with 70% ethanol, and dried.
  • Cerenkov counts are obtained on the dried pellets, and the pellets are resuspended and aliquots counted with scintillant.
  • the products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are then stained with ethidium bromide, dried and examined by autoradiography.
  • the hamster CHO cell line is obtained from the ATCC, and is maintained as recommended by the ATCC. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO 7 or lxlO 8 cells/ml.
  • the reaction components are the reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g) , and [ ⁇ - 32 P]dCTP (50 Ci) , mixed in a total volume of 50/1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Following preincubation, 0.5ml of the CHO cells are added to the mixture, the cells mixed and immediately transferred to the chilled 0.4cm electroporation cuvette. Electroporation is performed under the following conditions: 330V, 1000//F and infinite resistance.
  • the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent).
  • the radioactive supernatant is carefully removed and disposed of properly, and 1ml of 0.6% SDS (sodium dodecyl sulfate) , lOmM EDTA (pH 7.5) is added to the cell pellet.
  • SDS sodium dodecyl sulfate
  • lOmM EDTA pH 7.5
  • the extract is centrifuged for 20 minutes at top speed in a cold eppendorf microfuge. The supernatant is carefully withdrawn from the pellet (which is discarded in radioactive waste) , and the supernatant split between two new 1 * 5 ml microfuge tubes. These supernatants are extracted twice with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and 1/6 volume of 2-propanol; the pellets rinsed with 70% ethanol, and dried.
  • the pellets are resuspended in 18 1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0); the resuspended nucleic acid fraction is treated with ribonuclease A (2//1 of 10/g/ml stock solution), at 37 ⁇ C for 15 minutes, followed by the addition of 30/1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0). The solution is then extracted once with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and two volumes of ethanol; the pellets rinsed with 70% ethanol, and dried.
  • Cerenkov counts are then obtained on the dried pellets as described in section V. VII.
  • the human HeLa cell line is obtained from the ATCC, and is maintained as recommended by the ATCC. Prior to electroporation, the HeLa cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO 7 cells/ml.
  • the reaction components are the reverse transcriptase enzyme (1000 units) , the tRNA primer (5//g) , and [ ⁇ _32 P]dCTP (50//Ci) , mixed in a total volume of 50//1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Then 0.5ml of HeLa cells (5xl0 6 ) are added to the reaction components and the mixture is transferred into a chilled 0.4 cm electroporation cuvette (BioRad) . Electroporation is then performed at the following settings: 330V, 1000//F and infinite resistance.
  • oligo(dT) primer is desirably composed of oligomers of 12-18 bases of deoxyribothymidylic acid; 1 or 5/g of this oligonucleotide primer is added to reactions where indicated.
  • Cerenkov counts can be obtained on the dried pellets; the pellets can be resuspended (and aliquots counted with scintillant) , and the products electrophoresed on agarose/TAE gels. These gels are run with radiolabeled molecular weight standards, and the gels can be stained with EtBr (see Figures 5, 7, and 9) and dried and examined by autoradiography (see Figures 6, 8, and 10).
  • the reaction products are treated with SI nuclease to examine the nature of the polydeoxyribonucleotide product(s) .
  • SI nuclease will digest a double- stranded nucleic acid molecules to a limited extent, the enzyme works most efficiently on single-stranded nucleic acid substrates; thus polynucleotides which are single- stranded (or contain single-stranded regions) are digested to completion very quickly by SI nuclease treatment (Sambrook 1989) .
  • samples treated with SI nuclease when compared with untreated samples, show no appreciable degradation (Figure 10: compare lanes 2 & 3, and lanes 4 & 5) .
  • the SI nuclease treatment is performed on parallel samples before and after ribonuclease H treatment.
  • Ribonuclease H has the activity of digesting the RNA strand of a DNA- RNA heteroduplex. If the double-stranded cDNA product is a heteroduplex, then treatment of the material with ribonuclease H followed by treatment with SI nuclease will result in degradation of the material and loss of an autoradiographic signal in the higher molecular weight range of an agarose/TAE electrophoretic gel. When these experiments are done, the reaction products appear indistinguishable from the products of SI nuclease treatment alone. However, the experiments performed with RNase H and SI nuclease treatment will be repeated in order to rule out heteroduplex in vivo cDNA product.
  • the cells are treated as described in Sections V, VI and VII above.
  • the final pellets are rinsed with 70% ethanol, and dried. Cerenkov counts are obtained on the dried pellets, and the pellets are resuspended and aliquots are counted with scintillant.
  • the products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are stained with EtBr (see Figures 5, 7, and 9), dried and examined by autoradiography (see Figures 6, 8, and 10).
  • any second strand cDNA synthesis which occurs with this in vivo cDNA synthesis reaction is primed by the 3'-end of the first strand cDNA product.
  • RNase H activity acts through RNA template degradation to free the 3'-end of the first strand cDNA product and allow flanking sequences to become accessible to annealing by this 3'-end. This is a mechanism common in in vitro cDNA synthesis with this enzyme (Okayama, H. , et al. Recombinant DNA Methodology. Academic Press, Inc., pages 235-260 (1989)).
  • the in vivo cDNA reaction products are treated with SI nuclease and rendered blunt-ended with T4 DNA polymerase and high concentrations of dNTPs, prior to the addition of adapters or linker molecules. It is important that SI nuclease be diluted immediately before use, and that the concentration used is determined with small portions of the cDNA product prior to scale-up reactions.
  • the cDNA is resuspended in SI nuclease buffer: 200mM NaCl, 50mM NaOAc (pH 4.5), lmM ZnS0 4 , 0.5% glycerol and treated with approximately 100 units of diluted SI nuclease per ⁇ g of cDNA for 30 minutes at 37°C (Kimmel, A.R. , and S.L. Berger, Guide to Molecular Cloning Techniques , Academic Press, pages 328-329 (1987)). EDTA is added to 20mM to stop the reaction. The products are extracted twice with buffered phenol/chloroform, then once with chloroform.
  • the product is treated with T4 DNA polymerase in the presence of high levels of dNTPs to insure that the ends are blunted.
  • the cDNA product is suspended in T4 DNA polymerase buffer (50mM Tris-HCl (pH 8.3), 50mM NaCl, lOmM MgCl 2 , lOmM DTT) .
  • T4 DNA polymerase buffer 50mM Tris-HCl (pH 8.3), 50mM NaCl, lOmM MgCl 2 , lOmM DTT
  • a stock solution containing each of the deoxynucleoside triphosphates is added to achieve a 500//M final concentration.
  • Ten units of T4 DNA polymerase is then added to a final volume of 50//1, and the reaction is incubated at 37 ⁇ C for 30 minutes. The reaction is stopped with the addition of EDTA to 20mM.
  • the products are extracted, precipitated, and the pellet rinsed and dried, as described
  • Adapters or linkers can now be added to the cDNA product.
  • the use of annealed hemiphosphorylated adapters (Promega; only the 5' blunted end of the adapter is phosphorylated) is considered advantageous in that it allows cloning of the cDNA product after ligation of the adapters and elimination of the excess unligated adapters, without digestion of the cDNA insert to remove linker concatemers.
  • the step of removing the excess adapters, necessary to eliminate "linker library" construction can be utilized as well for sizing the cDNA prior to ligation into a vector.
  • annealed unphosphorylated linkers can be utilized and the excess unligated linkers can be removed simply by heating the ligation product briefly in the range of 60°C to 70°C. This melts the unligated sticky ends, and the ligated, linkered cDNA product can be separated away by column chromatography or gel electrophoresis.
  • the ligation reaction is performed as follows: to a tube is added 2//1 of 10X ligation buffer (666mM Tris-HCl (pH 7.6), lOOmM MgCl 2 , lOOmM DTT, 3mM ATP, lOmM spermidine-HCl , lOmM hexaminecobalt chloride, 2mg/ml bovine serum albumin) , hemiphosphorylated adapters, double-stranded, blunted cDNA, and 5 units of T4 DNA ligase in 20//1 total volume. The mixture is incubated overnight at 15 ⁇ C.
  • 10X ligation buffer 666mM Tris-HCl (pH 7.6), lOOmM MgCl 2 , lOOmM DTT, 3mM ATP, lOmM spermidine-HCl , lOmM hexaminecobalt chloride, 2mg/ml bovine serum albumin
  • the ligation of blunt ends can be enhanced by the addition of PEG 8000; however, polyethylene glycol inhibits phage packaging reactions, and so, should be removed completely prior to these -60- reactions.
  • the ligation reaction is then loaded on a 1ml bed volume Sephacryl S400 spin column (Promega) , which is centrifuged at 800xg to remove excess adapters and adapter dimers.
  • Alternative techniques which allow a more accurate size selection of cDNA product include agarose gel electrophoresis followed by capture and elution from cationic nitrocellulose (e.g. DEAE nitrocellulose) , or recovery directly from harvested sections of low melt agarose.
  • the cDNA is quantified using scintillation, and the molar ends calculated (based on quantity and average size) .
  • the cDNA inserts are now ready for ligation into the vector of choice (constrained somewhat by the size of the inserts and the compatibility of the sticky end) .
  • the ligation is performed as described above. With the use of hemiphosphorylated or unphosphorylated adapters, multiple insert cloning is unlikely, as only the vector has accessible phosphorylated ends.
  • the ligation reaction is then used for phage packaging, or diluted for chemical transformation or electroporation.
  • Colonies or phage which appear are initially screened with blue/white color selection (alpha complementation) , or with probes to abundant genes.
  • random colonies or plaques are examined for insert size using PCR with primers which flank the cloning site, or by restriction digest analysis.
  • libraries are constructed from cDNA made from CHO cells using the in vivo cDNA synthesis method.
  • One of the libraries is made from the tRNA Ht -primed in vivo cDNA product ( ⁇ 1.9kb) from CHO cells (see Figure 8, lanes 4 and 8) which is size- selected on an agarose gel, using the DEAE nitrocellulose (NA45, S&S) capture/elution procedure, prior to cloning.
  • Another library is made from the tRNA pol ⁇ -primed in vivo cDNA product from CHO cells, which is size selected for cDNA between 4-10 kb using agarose gel electrophoresis.
  • the cDNA product (-1.9 kb) which appears in CHO cells with the tRNA Mt primer may represent sequence- specific priming and synthesis from the conserved, moderately repetitive C-type and intracytoplasmic A-type particle (IAP) sequences found in all CHO cell lines examined (Anderson, K.P. et al . , Virol . 181:305-311 (1991)). These species have extensive homology to the genome of murine leukemia virus.
  • IAP intracytoplasmic A-type particle
  • Cellular reverse transcriptase cognate tRNA molecules, with modified ribonucleotide bases prime retroviral cDNA synthesis during viral replication. These molecules are not exclusively capable of priming in vivo cDNA synthesis, as evidenced by this invention and inferred from work previously done in vitro (Barat, C. et al . , Nucleic Acids Res. 19:751-757 (1991); Weiss, S. et al . , Gene 111:183-197 (1992); Kohlstaedt, L.A. , and T.A. Steitz, Proc. Natl . Acad. Sci . USA 89:9652-9656 (1992)).
  • fragments of synthetic tRNA molecules which lack base modifications are capable of annealing to RNA templates in vitro and directing in vitro cDNA synthesis from those templates (Weiss, S. et al . , Gene 111:183-197 (1992)).
  • oligonucleotide that is (a) capable of binding in vivo to an RNA template molecule and (b) acting as a primer for at least one reverse transcriptase enzyme such that synthesis of DNA complementary to that RNA template molecule occurs, is a legitimate primer for the in vivo cDNA synthesis of the present invention.
  • Experiments which both quantitatively and qualitatively assess the suitability of a prospective primer may be performed, e.g. , using Chinese hamster ovary (CHO) cells (see Example 1, above) .
  • the in vivo assays consist of introduction of [ ⁇ - 32 P]dCTP, putative primer, and Moloney murine leukemia virus reverse transcriptase into cells via electroporation.
  • Controls for assessing the suitability of primers for in vivo cDNA synthesis can include the following negative controls:
  • An actual primer test reaction can consist of:
  • a positive control reaction can consist of: 4) Electroporation of [ ⁇ _32 P]dCTP along with the modified tRNA pol ⁇ j primer and the reverse transcriptase enzyme.
  • the hamster CHO cell line is obtained from the ATCC, and is maintained as described in Example 1. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO 8 cells/ml.
  • the reaction components include the reverse transcriptase enzyme (1000 units) , the tRNA primer (5 g; or a molar equivalent of the candidate primer), and [ ⁇ - 32 P]dCTP (50/Ci) , mixed in a total volume of 50/1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes.
  • Electroporation is performed under the following conditions: 330V, 1000//F and infinite resistance. After electroporation, 1ml of warmed, C0 2 -equilibrated Ham's medium (GIBCO) is added to the cuvette, the mixture is transferred to a plastic tube (Falcon #2059) , and the mixture is then incubated for one hour at 37 ⁇ C.
  • the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) , and subjected to the extraction protocol described in Example 1, Section VI, and Cerenkov counts are obtained on the dried pellets.
  • the pellets are resuspended and aliquots are counted with scintillant.
  • the products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are then stained with ethidium bromide, dried and examined by autoradiography.
  • test primer may be priming cDNA synthesis.
  • the nature of the cDNA product(s) is very important and should be examined.
  • the second criteria is a qualitative assessment of the product and is determined initially from the pattern and density of the autoradiographic signal obtained from test aliquots run, in parallel with radiolabeled molecular weight standards, on the agarose/TAE gel. If the polynucleotide primer is designed to anneal to an RNA template of heterogeneous size, or to a binding site which is a heterogeneous distance from the 5'-end of the RNA template molecule, then one expects a heterogeneous population of cDNA products.
  • a specific cDNA product should be produced (compare, for example, Figure 8, lanes 4 and 5; and lanes 8 and 9) . Therefore, both qualitative and quantitative parameters should be examined to determine the efficiency of a prospective primer.
  • the present invention also provides for sequence- specific in vivo cDNA synthesis reactions using a sequence-specific primer which is introduced into cells in combination with a compatible reverse transcriptase enzyme and, if desired, modified deoxynucleotide triphosphate or deoxynucleotide triphosphate analogs.
  • sequence-specific primers can be synthesized as transcripts prepared from a DNA template or by chemical means (e.g. on solid phase supports) .
  • chemical synthesis using solid phase support The ability to chemically synthesize unmodified oligonucleotides, and 2'-0-methyloligoribonucleotides in high yields on solid phase permits the present invention to be readily adapted for use in preparative, analytical and therapeutical applications.
  • Synthetic techniques and reagents useful for modified oligoribonucleotide production provide a number of embodiments which could not be achieved if the RNA primers are produced in vitro from a DNA template. For example,
  • primers can then be fluorpphore- or hapten-conjugated, biotinylated, 32 P-labeled, or contain sequence elements such as restriction sites which will be copied and incorporated during second-strand cDNA synthesis following base-catalyzed hydrolysis of the original template (the 2'-0 _ Methyl primer will protect the annealed complementary portion of the original RNA template as well) .
  • sequence elements such as restriction sites which will be copied and incorporated during second-strand cDNA synthesis following base-catalyzed hydrolysis of the original template (the 2'-0 _ Methyl primer will protect the annealed complementary portion of the original RNA template as well) .
  • These modifications do not appear to disrupt the ability of the primers to associate with proteins (Sprout, B.S. et al . , (1989)), and the modified oligoribonucleotides anneal with the expected specificity.
  • the elimination of the 2'-OH group renders an RNA primer
  • sequence-specific DNA template One embodiment capable of producing the desired sequence-specific tRNA primer consists of in vitro transcription from a DNA template, generally as follows:
  • the initial template for the production of a expression cassette, encoding a sequence-specific tRNA primer can be the pUC18-T7tRNA Ht vector construct.
  • This construct encodes the tRNA wt molecule (see Figures 2B and 3A) .
  • the first of two primers will anneal to sequences which lie in the pUC18 vector 5' to the T7 promoter sequence (see Figure 4) and extend in the 3' direction toward the T7 promoter sequence.
  • a commercially available primer has been used in this manner in the amplification of the pUC18-T7tRNA pol ⁇ J expression cassette.
  • the second primer will desirably include bases at the 5'-end of the primer which are complementary to a sequence in the RNA template of the in vivo cDNA synthesis reaction (e.g. an RNA template of known sequence) , but are not complementary to the sequence of the initial DNA template (see Figure 4) .
  • the remaining bases in the primer will be complementary to bases in the DNA template which are a like distance from the precise 3'-end of the encoded tRNA template in the pUC18-T7tRNA Ht DNA vector, and extend in the 3' direction toward the T7 promoter sequence (i.e.
  • This cassette produces a tRNA spec1fjc molecule with a 3'-end which is complementary to both the 5'-sequence encoded in the second PCR primer, and the RNA target for the in vivo cDNA synthesis reaction (see Figure 4) .
  • PCR is performed in 20/1 volumes according to the following conditions: PCR buffer: 67mM Tris (pH 9.2 at 25°C), 16.6mM (NH 4 ) 2 S0 4 , 1.5mM MgCl 2 ; 50ng of each primer, approximately lxlO 8 molecules of the pUC18-tRNA Ht construct, and 250 M concentrations of each of the deoxynucleotide triphosphates.
  • the reaction mixtures are heated to 100 ⁇ C for 3 minutes, then cooled to 15 ⁇ C.
  • the tubes are centrifuged briefly to collect the contents, then 1 unit of Taq polymerase and a drop of mineral oil are added to each 20/1 reaction.
  • the reaction is performed in 40 cycles with the following regimen: 1 minute at 94 ⁇ C; then 1 minute at 55 ⁇ C.
  • the products are pooled from 5 reactions, blunted with the Klenow fragment, extracted to remove protein and traces of mineral oil, and EtOH precipitated using standard protocols.
  • the fragments are resuspended in lmM Tris- chloride (pH 8.0), O.lmM EDTA and an aliquot is examined on a 2% agarose/TAE gel with known size standards to verify size and to quantify.
  • Typical yields are expected to be approximately 500ng to l//g of product for each 20 1 reaction.
  • the tRNA speci ic molecule is then produced in an in vitro transcription reaction in a manner similar to that used for the production of tRNA polyU , above. Briefly, to RNase-free eppendorf tubes the following components are added at 25°C: 80mM Hepes-KOH (pH 7.5), 12mM MgCl 2 , 20mM DTT, 5mM dNTPs, 2mM spermidine; RNase-free dH 2 0; 50- 100/g/ml template DNA and 5/1 of [ ⁇ 3 P]ATP (30 Ci; 3000Ci/mmol; added in order to examine and quantify the products) .
  • reaction components are mixed, and the reaction is then initiated with the addition of T7 RNA polymerase enzyme reaction mix (to a final concentration of 1200-1800 U/ml) .
  • T7 RNA polymerase enzyme reaction mix to a final concentration of 1200-1800 U/ml
  • the tubes are then incubated at 37°C for 4 hours.
  • RNase-free DNase is then added to the reactions, and the digestion of template DNA allowed to proceed for 15-30 minutes; at this point, small aliquots of the reaction mixture can be removed in order to determine the efficiency of incorporation (This can be achieved by cold trichloroacetic acid precipitation of an aliquot of the reaction mixture in the presence of an excess of RNase-free carrier DNA.
  • the control for this experiment total counts, is done on unprecipitated material from the same reaction mixture) .
  • each of the reaction mixtures is then added 150 1 of RNase-free dH 2 0 and 20//1 of 3M NaOAc and the mixtures are extracted with an equal volume of phenol/CHCl 3 (pH 6.5), followed by CHC1 3 .
  • the products are precipitated with 2-propanol, the pellets rinsed, and the precipitate dried.
  • the primer product is resuspended in RNase-free dH 2 0, and an aliquot checked for size using autoradiographic exposure of a polyacryla ide/urea gel run with known size standards. The primer is purified and quantified prior to introduction into cells.
  • This approach allows direct amplification of DNA cassettes which are used for in vitro production of sequence-specific tRNA primers for use with the MoMuLV reverse transcriptase enzyme.
  • the technique utilizes a single sequence-specific DNA primer, a 5' universal primer and a tRNA wt DNA template for cassette amplification.
  • sequence-specific primers for use in the in vivo cDNA reactions enables an investigator to take a real time "snapshot" of the transcription patterns which exist in a target cell or tissue.
  • Care in the preparation of the target cells for this analytical use of the in vivo cDNA synthesis technology is, perhaps, more important than for simple preparative use (e.g. cDNA library construction) , and real assessment of the type of RNA primer, the mode of primer-reverse transcriptase (and, perhaps modified deoxynucleotide triphosphate, or analog) and delivery should be carefully considered.
  • Many alternatives exist for delivering the primer/enzyme complex and a representative selection have been outlined above.
  • One technique for the manufacture of a DNA cassette which allows in vitro production of the sequence-specific primer is outlined in Example 3, above.
  • the primer product is resuspended in RNase-free dH 2 0, and an aliquot checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards.
  • the primer is purified and quantified prior to introduction into cells.
  • RNA primer on solid phase support, generally as described in Example 3, Section II.
  • This approach allows the direct synthesis of the sequence-specific primer, as well as providing an opportunity to incorporate modified stable ribonucleic acid analogs which would be useful for detection, purification and/or modification of the cDNA products (e.g. 32 P-labeling, biotinylation, and/or sequence incorporation, respectively) .
  • the cells are prepared in a manner which allows a minimum of perturbation to the desired conditions, and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue.
  • the sequence-specific primer, reverse transcriptase enzyme, and [ ⁇ -32 P]dCTP are incubated together briefly in vitro (as described in Example 1, above) , prior to addition to the cells or tissue. Following introduction to the cells or tissue, the cells or tissue is incubated under conditions which permit the synthesis of DNA molecule(s) which are complementary to the specific RNA template.
  • the controls for this reaction include the following: 1) Delivery of [ ⁇ 32 P]dCTP alone. 2) Delivery of [ ⁇ -32 P]dCTP along with the reverse transcriptase. 3) Delivery of [ ⁇ -32 P]dCTP along with in vitro- transcribed "wild type" primer and the appropriate reverse transcriptase enzyme.
  • the cDNA products can be qualitatively examined by autoradiographic analysis of gels run with appropriate radiolabeled size standards.
  • specific products can be quantified by scanning autoradiographs of analytical electrophoretic gels or by liquid scintillation counting of bands excised from preparative electrophoretic gels.
  • the PCR can be used to detect any cDNA which is produced from unprocessed RNA template.
  • sequence-specific primers for use in the in vivo cDNA reactions also enables one to use a real time "snapshot" of the target cell transcription patterns to detect differential transcription patterns and transcript levels, which are associated with numerous disease states. It is desirable from both a diagnostic and research perspective to identify the levels of various oncogene transcripts, in relation to the levels of an internal control transcript (e.g. a "housekeeping" gene) .
  • PCR has been used for this purpose in the past and is, at best, unreliable and biased due to problems which occur during amplification (Gilliland, G. , et al . (1990)).
  • probe and control primer products are resuspended in RNase-free dH 2 0, and aliquots are checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards.
  • the primers are purified and quantified prior to co-introduction into the target cells or tissues.
  • the cells are prepared in a manner which allows a minimum of perturbation to the desired conditions, and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue.
  • an oncogene sequence-specific primer and an actin sequence-specific primer are utilized as probe primer and control, respectively.
  • the cells are prepared in a manner which allows a minimum of perturbation to the desired conditions and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue.
  • the sequence-specific primer, reverse transcriptase enzyme, and [ ⁇ -32 P]dCTP are incubated together briefly in vitro, as described in Example 1, above, prior to addition to the cells or tissue. Following introduction to the cells or tissue, the cells or tissue is incubated under conditions which permit the synthesis of DNA molecule(s) which are complementary to the specific RNA template.
  • the controls for this reaction might include the following:
  • the cDNA products can be qualitatively examined by ethidium stained gels (see Figure 7, lanes 4 and 8), or autoradiographic analysis of gels run with appropriate radiolabeled size standards (see Figure 8, lanes 4 and 8) .
  • the level of incorporation can be determined by measurement of Cerenkov counts, or, following resuspension, by liquid scintillation counting of aliquots.
  • [ ⁇ -32 P]dNTPs a biotinylated, sequence-specific tRNA primer, the reverse transcriptase, and one of the four (4) dideoxynucleotide triphosphate chain-terminating molecules into the target cell.
  • four separate introductions are performed, each introducing a different chain-terminating dideoxynucleotide base.
  • the cells are then incubated for the time, and in a manner which allows incorporation.
  • the nested cDNA products are extracted and concentrated using, e.g.
  • an avidin-coated polystyrene (Baxter Healthcare, Mundelein, IL) or -77- streptavidin-1inked bead, and eluted from the bead by heating in sequencing gel loading buffer just prior to loading a sequencing gel.
  • the sequence can then be determined by autoradiography.
  • these techniques permit a direct approach to determining the 5'-sequence of clones where information concerning the 3'-sequence is available.
  • the choice of a specific 18 base primer sequence will permit specific sequencing in vivo without the time and difficulty of cloning the cDNA product.
  • deoxynucleotide analogs into the in vivo cDNA product allows the preparation and recovery of biotinylated cDNA which can be useful in a number of important areas.
  • Specific biotinylated deoxynucleotide analogs are appropriate for specific experiments, and many candidate substrates exist (Klevan, L. , and Gebeyehu, G. , Meth. Enzymol . 154:561-577 (1987)).
  • the following Example utilizes one approach and should not be construed as defining or limiting alternative methods or techniques for biotinylation of the in vivo cDNA product.
  • the hamster CHO cell line is obtained from the ATCC, and maintained as recommended. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted. rinsed in phosphate buffered saline and resuspended in phosphate buffered saline at lxlO 8 cells/ml.
  • the reaction components are reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g; can be either sequence-specific, or a general primer, e.g.
  • tRNA polyU tRNA polyU
  • bio-11-dUTP 0.03mM or 0.3mM final
  • BBL reverse transcription buffer
  • DTT lOmM
  • Electroporation is performed under the following conditions: 330 volts (V) , 1000 microfarads (//F) and infinite resistance.
  • the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) , and the cDNA product is recovered as described in Example 1.
  • the biotinylated cDNA pellets are resuspended, and the products electrophoresed on agarose/TAE gels. These gels can be run with biotinylated molecular weight standards, and the gels can be transferred and the Southern blot examined using a streptavidin/alkaline phosphatase conjugate system (e.g. the BluGENE System, Bethesda Research Laboratories, Gaithersburg, MD) .
  • a streptavidin/alkaline phosphatase conjugate system e.g. the BluGENE System, Bethesda Research Laboratories, Gaithersburg, MD
  • the products can be purified and extracted from preparative agarose gels and used for probes in electron microscopy (using streptavidin-gold) , for in situ hybridization (using streptavidin/alkaline phosphatase detection system (Chan, et al . (1985)), subtractive library construction, as well as other preparative, analytical and therapeutic purposes.
  • the ability to increase the relative frequency of induction-specific, or cell or tissue-specific messages prior to the construction and screening of a cDNA library, is of great strategic benefit, and often the only hope of identification and recovery of genes which are represented by mRNAs of low abundance (Hedrick, S. et al . , Nature 308:149 (1984)).
  • the previously known subtraction techniques utilizes a separation strategy whereby single-stranded cDNA (produced from mRNA selected from the desired cells, tissues or conditions) is annealed to an excess of mRNA extracted from alternate cells, tissues or conditions.
  • the heteroduplex molecules are separated from the single-stranded cDNA (and mRNA) using hydroxylapatite, or other selective matrices.
  • the ability to incorporate deoxynucleotide analogs into the in vivo cDNA product allows the preparation and recovery of biotinylated cDNA.
  • biotinylated cDNA product of this in vivo incorporation is in subtractive library construction.
  • This alternative technique of library construction is superior to the prior art, in that both the desired template, and the subtracting template molecules are cDNA, allowing a stability during the hybridization and selection process not previously possible.
  • the cDNA product is produced from a smaller initial number of cells, with a fidelity and processivity which may exceed the quality of products produced in vitro .
  • This technique relies on the hybridization of biotinylated second-strand cDNA product of one cell population with the cDNA product of first- strand syntheses of the alternately grown or treated cells. Therefore, parameters must be evaluated which yield efficient second-strand products which are biotinylated.
  • Choices in reverse transcriptase enzymes and the like are important, and if the quantity of second-strand product is not sufficient, non-biotinylated in vivo cDNA synthesis may be coupled with second-strand synthesis in vitro (with biotinylated analog incorporation) to augment the amount of this material.
  • the incorporation reaction can be performed as described in Examples 3 or 8, or by alternate means.
  • the biotinylated cDNA is produced in cells or tissue which is grown under conditions other than those of the desired cDNA library.
  • the extracted and isolated biotinylated cDNA product is treated with SI nuclease to cleave any hairpin structure(s) which might link first-strand product from second-strand product; separated from any contaminating sheared genomic DNA by column chromatography or gel electrophoresis, and annealed to approximately 1/30 the quantity of non-biotinylated cDNA product which was produced from the desired cell or tissues, or from cells or tissues grown under alternate, desired conditions (and similarly separated from genomic DNA contaminants) .
  • Routine hybridization conditions are: 120mM NaH 2 P0 4 (pH 6.8), 820mM NaCl, lmM EDTA, 0.1% SDS, with the final DNA concentration at approximately 5mg/ml.
  • the reaction mixture is heated to 90°C for 5 minutes, then maintained at 65 ⁇ C for 12-18 hours.
  • the reaction is diluted in phosphate buffer (120mM NaH 2 P0 4 , pH 6.8), and the biotinylated cDNA, with annealed common cDNA sequences, is isolated on a streptavidin column.
  • the flow-through cDNA fraction is concentrated by ethanol precipitation and cloned using accepted methods and techniques (Sambrook, (1989)).
  • a human immunodeficiency virus, type I sequence- specific primer for use in the present in vivo cDNA synthesis invention is made with an appropriate HIV- specific sequence.
  • One such PCR primer is prepared, based on the sequence of the SK69 primer (Ou, C-Y et al . , Science 239:295-297 (1988); Zack, J.A. et al . , Cell 61:213-222 (1990)) corresponding to the env region of HIV, as follows (SEQ ID NO. 9):
  • a PCR primer can be based on the M666 primer of Watson & Wilburn (1992) , corresponding to the U 3 region of LAV-1 BRU , as follows (SEQ ID NO. 10) : 5'-GGGGAGTGGC GAGCCCTCTT GAACCCGGGA CCTCTCGCAC CCC
  • the purified, examined and quantitated primer is then introduced into cytokine-activated (Poli, G. and A.S. Fauci, AIDS Res. Hum. Retroviruses 8:191 (1992)) lymphocytes (either fractionated, or unfractionated) utilizing appropriate technique(s) (among them, electroporation and cationic lipid-mediated delivery) , along with MoMuLV reverse transcriptase enzyme, and fluorescent deoxynucleotide triphosphate analog(s) .
  • the cells are incubated under conditions and for a time necessary to produce cDNA to any existing HIV-specific RNA template present in the target cells.
  • a control reaction can be performed on a similar, uninfected cell population It may prove advantageous that many of the fluorophore-conjugated nucleotide analogs undergo a spectral shift upon polymerization. This may allow discrimination between cells in which template-directed cDNA synthesis has occurred, and the background cell population(s) .
  • the cells are washed and then, if desired, incubated on ice for 30 minutes with an antibody directed to the CD4 molecule (e.g. OKT4 from the ATCC) , washed and incubated with FITC-labeled F(ab') 2 goat anti-mouse IgG.
  • the cells are then washed again, prepared and screened by Fluorescence Activated Cell Sorting (FACS) using parameters (e.g. gating for cell size, fluorescence and threshold spectra) necessary to excite and screen for the incorporated fluorescent tag(s) .
  • FACS Fluorescence Activated Cell Sorting
  • Appropriate parameters can be determined using the negative control cell populations.
  • the cells are grown as monolayer culture in 60mm dishes to 60-90% confluence under conditions to maximize transcript populations (e.g. adding interferon to increase the transcription of interferon-induced genes) .
  • the reaction components the reverse transcriptase enzyme (1000U), the tRNA primer (2-5 ⁇ g) , and [ ⁇ - 32 P]dCTP (50 ⁇ Ci) , mixed in a total volume of 50-100 ⁇ l in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes.
  • DOTMA dimethyl-dioctadecyl ammonium bromide
  • DOPE liposomes 1:1 (w/w of DOTMA (N-(1,2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride) :DOPE liposomes, and the mixture is incubated at room temperature for 5-30 minutes. An additional 0.5ml of serum-free medium is then added to the mixture. The medium is aspirated off the cell monolayer and the monolayer is rinsed twice with serum-free medium. The reaction mixture is added dropwise to the dish and the dish is immediately rocked to evenly coat the monolayer with the reaction solution.
  • DOTMA N-(1,2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride
  • cDNA product can be immediately harvested after the 2 hour incubation period following transfer of the radiolabeled reactants to radioactive waste, and rising the monolayer 2X with phosphate buffered saline.
  • the medium is removed from the monolayer, and replaced with 1.5ml of cold medium.
  • the cells are scraped from the dish with a disposable policeman and transferred to microfuge tubes.
  • the remaining cells are rinsed from the dish with 0.5ml of medium which is combined with the cells.
  • the cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) .
  • the radioactive supernatant is carefully removed and discarded properly.
  • a Hirt extraction is carried out on the cell pellets to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions. The cDNA products are then precipitated using standard techniques.
  • the cells are grown in suspension culture and split at regular intervals prior to protocol to insure optimal viability.
  • the reaction components the reverse transcriptase enzyme (1000U) , the tRNA primer (l-10 ⁇ g) , and [ ⁇ - 32 P]dCTP (50 ⁇ Ci) , mixed in a total volume of 50- lOO ⁇ l in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes.
  • DOSPA 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethy1- 1-propanaminiumtrifluoroacetate
  • DDAB dimethyl-dioctadecyl ammonium bromide
  • DOTMA N-(1,2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride
  • the medium is aspirated from the cell pellet.
  • the reaction mixture is added to the tube and the tube is immediately flicked to gently resuspend the cell pellet in the reaction solution.
  • the cells are periodically gently flicked to resuspend the cells during a 1 hour incubation period in a 37°C C0 2 incubator.
  • Three (3) mis of prewarmed and C0 2 -equilibrated medium containing serum is then added to the cells and the cells are transferred to 60mm dishes (Falcon) , and the dishes are returned to the 37"C incubator for 1-4 hours.
  • cDNA product can be immediately harvested after the 1 hour incubation period following transfer of the radiolabeled reactants to radioactive waste, and rinsing the cell pellet 2x with phosphate buffered saline.
  • the cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) .
  • the radioactive supernatant is carefully removed and discarded properly.
  • a Hirt extraction is carried out on the cell pellet to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions.
  • the cDNA products are then precipitated using standard techniques.
  • mice are obtained from Jackson Labs, maintained under pathogen-free conditions, and provided food and water ad lib.
  • the in vivo cDNA reaction components reverse transcriptase enzyme (1000U-5000U) , a IgG heavy chain-specific tRNA primer (2-20 ⁇ g) , and [ ⁇ - 32 P]dCTP (50-500) ⁇ Ci);, are mixed in a total volume of 200 ⁇ l in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes.
  • reverse transcriptase enzyme 1000U-5000U
  • IgG heavy chain-specific tRNA primer 2-20 ⁇ g
  • [ ⁇ - 32 P]dCTP 50-500
  • DOTMA N- (1,2,3-dioleyloxy)propyl)-N,N,N-trimethy1ammonium chloride
  • D0PE liposomes D0PE liposomes
  • the 0.5ml mixture is slowly injected either into the intraperitoneal cavity or into the tail veins of the mice.
  • the mice are sacrificed 4-24 hours later.
  • the heart, liver, brain, pancreas, and lymph nodes and spleen are removed and weighed. Tissue sections are counted with scintillant and the counts corrected for the weight of the tissue samples. The normalized counts are high in both the spleen and lymph node tissue sections.
  • the tissue is gently disrupted, debris is removed and the cells are carried through a Hirt- extraction.
  • the episomal cDNA products are extracted with phenol/chloroform followed by chloroform.
  • the cDNA products are then precipitated using standard techniques.
  • the cDNA products are fractionated (e.g. electrophoresis through a buffered agarose matrix) , and visualized by autoradiography.
  • fluorophore- or hapten-conjugated deoxynucleotide analogs can be delivered (or the primer can be labeled) , and the location of the in vivo cDNA products determined by fluorescence of tissue sections (or extracted products) , or by secondary reagents directed at the incorporated analogs.
  • a retrovirus reverse transcriptase enzyme complexed with its modified cognate tRNA primer (having a sequence which includes a number of polyuridylic acid residues 5' to a 3'-terminal region containing a specific polynucleotide sequence) will position the enzyme complex on RNA template molecules at the junctional region proximal to the polyadenylic acid stretch present on mature messenger RNAs such that a snapshot of members containing a complementary junctional sequence will be converted to cDNA product.
  • an initial specific polynucleotide template primer molecule may also be used for this purpose, and may prove advantageous in that (1) when an initial specific RNA template molecule is included in the protocol the 5'-region of the initial template molecule can be designated to encode a much more extensive pattern of bases. This will provide an energetically more favorable and potentially more discriminating annealing platform from which priming of cellular messages can occur; and, (2) sequence elements can be included in this initial template primer molecule which will increase the efficiency of initiation of cDNA synthesis.
  • the pattern may be diagnostic for cellular perturbation (e.g. transformation or infection) , or change in response to an applied stimulus. Changes may be characterized solely by changes in the density of various cDNA products which make up the pattern.
  • This pattern technology will be a powerful tool both for diagnostic purposes and for following developmental changes or lineage commitment in cell populations. Fluorophore- or hapten-conjugated deoxynucleoside triphosphate analogs can be used to enhance detection of the in vivo cDNA products. Alternatively, nuclease- resistant (e.g. 2'-0-methyl) and labeled primers may be used for the reactions.
  • RNA template primer(s) are introduced into the cells by any of a variety of means (see Examples 1, 11, and 13), or the cells are infected with a defective retrovirus element which includes a template primer molecule whose cDNA product is complementary to the annealing region of the final target polynucleotide molecule (and has complexed within the virus the cognate modified tRNA primer which anneals in the 3'-region of this initial template and a suitable reverse transcriptase enzyme) , the primer-directed conversion of RNA template to cDNA product(s) effectively removes the transcript(s) from the processes required for normal cellular use or expression.
  • enzymatic conversion as opposed to simple hybridization (e.g. antisense nucleotides) or cleavage step (e.g. ribozymes) results in the efficient and stable conversion of template to inert molecular form.
  • This provides an additional advantage, for the products of the antisense reaction can be readily detected, quantified and evaluated.
  • introduction of a recombinant, defective, defined-tropic virion or particle which contains a VCE template primer molecule with the associated reverse transcriptase enzyme -90- or complex thereof, together with a promoter element (inducible or otherwise) to drive the antisense expression of an included heterologous gene will provide a vehicle for sustained gene therapy.
  • the initial steps of this strategy can be utilized by including into an initial RNA template sequences which will be converted to cDNA; then, as in proviral replication, the initial RNA template will be hydrolyzed by the reverse transcriptase-associated RNase H activity, and the remaining single-stranded cDNA will anneal to a polynucleotide template which has a sequence which is complementary to the 3'-end of this initial cDNA molecule. cDNA synthesis will then be resumed and the second template will be covalently joined to the first cDNA transcript within the viable cell. As shown in Figure 12, this strategy allows the intracellular cloning of any available RNA template molecule with the inclusion of desired vector sequences.
  • a region of polyadenylic acid residues at the 5'-end of the first RNA template will result in a polythymidylie acid 3'-region in the initial cDNA product which will anneal to any available polyadenylated messenger RNAs. This allows the in vivo synthesis and construction of a cDNA library.
  • a specific sequence can be included in the 5'-end of this initial RNA template in order to prime cDNA synthesis from specific transcripts.
  • the choice of length and sequence of any polypurine tract is important to preclude the initiation of strand synthesis prior to strand transfer. For the same reason, the choice of the reverse transcriptase enzyme may be important.
  • the reaction components reverse transcriptase enzyme (1000U) , a modified tRNA primer (l-10 ⁇ g) , and [ ⁇ - 32 P]dCTP (50 ⁇ Ci) , are mixed in a total volume of 50- lOO ⁇ l in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes.
  • the Vector Control Element (VCE) RNA template primer molecule is then added (in an amount dependent upon the size of the template segment) in an additional 30-50//1 of buffer, and the mixture is then incubated at room temperature for an additional 5-10 minutes. This mixture is then electroporated or otherwise introduced into viable eukaryotic cells and the cells are incubated for 1 hour under conditions which allow in vivo cDNA synthesis to occur.
  • VCE Vector Control Element
  • the modified tRNA primer may be annealed to the VCE RNA template primer molecule prior to the addition of the reverse transcriptase enzyme.
  • the reverse transcriptase is allowed to form a complex with the modified tRNA primer prior to the addition of the RNA template molecule.
  • the VCE RNA template primer will have a first sequence to which the modified tRNA primer will anneal and initiate cDNA synthesis, and includes a polynucleotide sequence at the 5'-end which is designed to be complementary and anneals to a 3'-region of a second RNA template molecule.
  • the VCE RNA template primer eliminates this constraint. This relaxation in size of the primer complementary region allows very efficient secondary priming of RNA templates with specifically designed single-stranded cDNA primers.
  • encoded in this initial RNA (GE or VCE) template primer may be one or more of the following: a promoter operatively linked to a gene which confers resistance for biological selection; cre/lox sites, a polycloning site containing restriction enzyme recognition sites; an origin of replication for procaryotic or eukaryotic replication; a promoter which is operatively linked to any cDNA which is inserted 3' to the promoter sequence (or a promoter which is operatively linked to produce an "antisense" message to any cDNA which is inserted) ; transcription enhancer or tissue- specific control elements; and regions which encode products which aid in the detection or recovery of product produced from an inserted sequence (e.g.
  • the cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) . The radioactive supernatant is carefully removed and discarded properly.
  • a Hirt extraction is carried out on the cell pellet to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions. The cDNA products are then precipitated using standard techniques.
  • the cDNA products are resuspended and may be subject to directed second-strand cDNA synthesis or other in vitro modification(s) .
  • the final products are ligated under conditions which favor intramolecular ligation (dilute conditions) .
  • the cDNA products are then transformed into bacteria and plated out under standard conditions which favor biological selection for the resistance conferred by the encoded genes, or gene(s) encoded by the VCE RNA template primer molecules.
  • Uridine and deoxyuridine triphosphate conjugates of haptens e.g. DNP or fluorophores (e.g. fluorescein-12-, tetramethylrhodamine-5-, Texas Red-5-, Cascade Blue-7-,
  • the deoxyuridine triphosphate analogs can be incorporated into the cDNA during in vivo cDNA synthesis.
  • each fluorophore analog has spectral properties (both excitation and emission) which are specific to the fluorophore group and the coupling, and there may be differences in the cytotoxicity or cell inducing properties associated with the various analogs.
  • each has a membrane partitioning coefficient which may be important in various applications. This latter parameter will affect the substrate availability within the cell or tissue, contribute to the background fluorescence following enzymatic incorporation, and may well limit the choices in the delivery systems.
  • Use of these analogs, or the "caged" analogs described below, in conjunction with a variety of delivery systems allows the in vivo incorporation of cDNA products within cells containing detectable levels of RNA template molecules. This delivery can be to individual cells and tissues, or the components can be introduced systemically.
  • Sequence-specific primer molecules allow transcript discrimination, and the combined use of radiolabeled primer(s) (or primer(s) labeled with a group which absorbs or emits at a different wavelength) and a second primer(s) introduced with the detectable nucleotide analogs which are incorporated during in vivo cDNA synthesis, will allow dual labeling or differential labeling of cells and tissues.
  • In vivo labeled cDNA products may be detected by any number of means including: FACS analysis of cells; tissue- and animal-sectioning coupled with autoradiography or fluorescence examination; use and detection of secondary reagents directed at incorporated nucleotide analogs; or a combination of these approaches (e.g.
  • Caged nucleotides e.g. the (2-nitrophenyl)-ethyl ester of dATP, Molecular Probes, Inc., Eugene, Oregon
  • caged nucleotide analogs which are labeled in conjunction with the present cDNA synthesis invention will allow light-directed cDNA synthesis and detection of the resultant cDNA product(s) .
  • Replication-defective retroviruses are used as infective vectors to introduce genes into eukaryotic cells.
  • sequences containing the gene(s) of interest are packaged into cell lines which express trans-acting functions necessary and lacking in the proviral template.
  • the packaged recombinant template contains obligate cis-acting sequences including 5'- packaging signals ( ⁇ ) , direct repeats (DR) , as well as subregions ( ⁇ + ) within the gag gene (Armentano, et al . , J. Virol . , 61:1647 (1987); Bender, et al . , J. Virol . , 61:1639 (1987); Adam and Miller, J. Virol .
  • Heterologous cellular transcripts which may be packaged by a cell line expressing trans-acting functions can be effectively identified and removed by treating the cells with a packaging signal-specific primer sequence (using a modified tRNA primer or an initial VCE RNA template primer) ,. along with a reverse transcriptase enzyme or complex thereof.
  • a primer sequence can be designed to discriminate between the intended target packaging signal, and fortuitous contaminating cellular sequences.
  • RNA templates make possible the in vivo conversion of RNA templates into a proviral form which can be integrated or maintained as an episomal element.
  • VCE Vector Control Element
  • Inclusion of promoter elements, centromere elements, telomere elements, origins of replication, as well as other control elements will allow the copy number of an episomal element to be controlled providing long-term meiotic and mitotic stability.
  • EXAMPLE 20 Inclusion of components to Enhance cDNA Synthesis
  • Retroviral nucleocapsid proteins as well as other retroviral proteins, have been shown to be significant in the dimerization and packaging of retroviral genomes and may be expected to be useful to optimize efficiency in the present methods as well

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Abstract

Methods and compositions for synthesizing cDNA are disclosed, wherein a synthetic polynucleotide template primer molecule is utilized.

Description

Description Methods and Compositions for cDNA Synthesis
Technical Field
The present invention relates to methods and compositions for DNA synthesis, and, more particularly, for the synthesis of complementary DNA.
Background of the Invention
The present invention is a tool for molecular biology. An introduction to the nomenclature of molecular biology, the structure of DNA, RNA and proteins and the interrelationships between these molecules, is provided in Chapter 4, Synthesis of Proteins and Nucleic Acids of Darnell et al . , Molecular Cell Biology. Scientific American Books (1989) . A more detailed treatment of these issues is set forth in the full text of Darnell et al . , (1989) and in Lewin, Genes IV. Oxford University Press (1990) .
Hereditary information is encoded in the genes of an organism. Genes are composed of polymers of nucleic acids. In higher organisms this nucleic acid is deoxyribonucleic acid (DNA) . DNA is composed of a series of four nucleotide bases; the hereditary information carried by a gene is encoded by the specific sequence of nucleotide bases in the DNA molecule. The genetic information within structural genes encodes proteins; the sequence and structure (and therefore function) of a particular protein is determined by the order of the nucleotide bases within the gene that encodes that protein. Proteins determine an organism's identity; from cellular structures to the organism's response to its environment. Thus, the genes that encode these proteins determine an organism's identity. The information encoded within a structural gene is "expressed" by a cell through the processes of transcription and translation. Transcription results in the production of an intermediate carrier of the genetic code, termed messenger RNA (mRNA) . Messenger RNA is effectively a copy of the gene; it is a polymer of ribonucleic acid (hence "RNA") rather than of deoxyribonucleic acid.
In eukaryotic organisms (which are generally more complex organisms than bacteria) , genes are made up of coding regions (termed "exons") and non-coding regions (termed "introns") . Exons directly encode the protein sequence of the gene. Introns may be very large and there may be a large number of intron sequences within a particular gene. The role of the non-coding intron sequences is unclear. However, there is evidence that these intervening sequences serve at least three critical purposes: first, they allow a cell to produce a nascent RNA transcript which can be spliced in several ways to generate a number of different proteins; second, they allow the inclusion of control elements within the intron regions which enhance the regulation of gene expression; and three, they relegate discrete portions or cassettes of protein sequence to exon units which can be more easily shuffled during the course of evolution and therefore facilitate the development of new proteins which may ultimately enhance the survival of the species. The transcription process involves the formation of an mRNA copy of the entire gene. That is, the mRNA produced by the transcription process contains a copy of both the non-coding intron sequences and the protein- encoding exon sequences. Thus the mRNA first produced by transcription is the same length as the gene from which it was copied. Subsequently, this immature mRNA undergoes a processing stage during which the non-coding intron sequences are spliced out. The resulting processed mRNA molecules thus contain only the information required to encode the protein (i.e. they contain copies of only the joined exon sequences) . These processed mRNA molecules are thus considerably shorter in length than the "genomic sequence" (the gene exons and introns as they exist in the chromosome) from which the mRNA was initially copied. The processed mRNA is also modified at this stage to include a polyriboadenylic acid, poly(A) , tail at one end of the molecule (the 3'- end) and a "cap" structure at the other end of the molecule (the 5'-end) (standard nomenclature assigns one end of DNA and RNA molecules as the 5'-end and the other as the 3'-end, according to the terminal chemical groupings of the molecule) . An mRNA molecule that has been processed to remove introns and has a 5'-cap and a 3'-poly(A) tail is termed a "mature" mRNA molecule. A greatly simplified diagram of the transcription process, illustrating removal of the non-coding intron sequences is shown in Figure 1.
The step of converting the information carried by the mature messenger RNA into a protein is termed translation. Translation is the final step of the means by which the information encoded by the nucleotide sequence within a structural gene is converted into a specific protein composed of a sequence of amino acids.
The cloning of genes became possible in the 1970's. In early experiments, small genes were cloned from bacteria and phage (bacterial viruses) . Since that time advances in molecular biology and genetic engineering have developed at an extraordinary rate, such that the sequence of the entire human genome is now being determined. Despite rapid advances in the technology of this field, a number of limitations are still apparent. One of these is the difficulty of cloning very large structural genes.
The size of a gene is measured in the number of nucleotide bases that it contains, usually expressed in terms of thousands of bases (kilobases or Kb) . Although there are several examples of larger genes, the total coding sequence of most structural genes (the exons) typically totals 1-10 Kb. However, the presence of multiple large intron sequences between the exon segments means that at the genomic level these genes are spread out over a much larger area, frequently spanning tens or even hundreds of kilobases. Present gene cloning vectors such as YACs (Yeast Artificial Chromosomes) allow the cloning of very large (100-300 Kb) genomic segments; however, these genomic inserts include the noncoding intron sequences, which precludes the expression of protein in an artificial system. Expression from a partial genetic sequence, or sequence containing introns, results in the production of a nonfunctional, truncated protein, or, when the sequence for the 5' translation start site is missing, results in expression of a unrelated garbled protein sequence. Therefore, even if a partial gene may be identified through a screening process, it is then necessary to recover the remaining portions of the gene. This can be an extremely complicated process. If the gene contains many intron sequences, and is thus large, years of effort can be expended in attempting to recover the remaining pieces of the gene. Additional effort may then be required to determine the relative order of the gene fragments and to distinguish exon from intron sequences. The ability to clone a gene as a contiguous protein coding cassette is particularly important where identification of the gene is achieved by means of a detection technique which relies on production of the protein in a recombinant bacterial or viral system and "screening" for the function or structure of the desired protein — a common technique of detecting cloned genes.
To clone structural genes, molecular biologists have taken advantage of the cellular mRNA processing function described above whereby intron sequences are spliced out of the immature mRNA to produce a mature mRNA that is considerably smaller that the original gene. By converting the mature mRNA molecule back into a DNA molecule (hence the term, "reverse transcription") , one can obtain the original coding sequence (the exons) without the extraneous intron sequences. Such a DNA molecule is termed a complementary DNA because it is complementary to the mRNA molecule from which it was derived. Complementary DNA (cDNA) synthesis is the preferred technique for gene cloning because it results in the recovery of the desired gene in a relatively small, contiguous protein coding cassette amenable to recombinant protein production. An additional and important use of cDNA technology is to identify those genes that are being expressed by a cell at a particular time. Gene expression requires substantial energy expenditure on the part of the cell, and mRNA molecules are designed to be short-lived "protein requests;" therefore, with a few exceptions
(notably in the egg during development) , only those genes that code for proteins which are immediately needed are transcribed into mRNA. By making cDNA copies of the existing mRNA population in a cell, and cloning the cDNAs produced, researchers are able to produce a cDNA library from the genes which were being expressed at that time. Researchers can thus determine specifically which genes are expressed in a given tissue type, at a given stage of development, or in response to an applied stimulus. Complementary DNA clones are extremely important in both research and industry. Research requires expression of the cloned gene in order to determine the protein's function and structure. In addition, large amounts of protein are required for both X-ray crystallographic structure analysis, and for the production of polyclonal or monoclonal antibodies which are indispensable for following small amounts of the protein through research protocols, and in determining the location of the protein in the cell.
Bacteria are commonly used as hosts in which a cloned gene is expressed. The genes of prokaryotes, including bacteria, do not contain introns, and so these cells do not have the splicing machinery necessary to process immature mRNA into a mature mRNA that can be translated into a functional protein. Genomic clones of eukaryotic genes (i.e., containing introns and exons) can not be expressed in a bacterial host, whereas a cDNA copy of the same gene can be expressed - either in prokaryotes or eukaryotes. Thus, cDNA clones are routinely used for large scale protein production. This artificial protein expression is termed "recombinant protein" production and is an increasingly common way of producing many of the pharmaceuticals which for years were accessible in small amounts by tedious extraction from other animal's tissues.
Techniques presently used for cDNA synthesis are reviewed in Berger and Kimmel, Guide to Molecular Cloning Techniques in Methods in Enzv ology Volume 152, Academic Press Inc. (1987), in Sambrook et al . , Molecular Cloning. Cold Spring Harbor Laboratory Press (1989), Okayama, H. , et al . , Meth. Enzymol . 159:3-27 (1987), Van Gelder, et a . , Proc. Natl . Acad. Sci . USA 87:1663-1667 (1990); and Embleton, M.J., et al . , Nucleic Acids Res. 20:3831-3837 -1-
(1992) . A review of mRNA isolation techniques is presented in Chapters 7 and 8 of Sambrook et al . (1989). Isolation of mRNA is a long, tedious process with a number of technically difficult steps. In summary, a typical procedure for isolating mRNA from a cell requires (1) disruption of cells to release cellular contents, (2) isolation of total RNA from the cell, (3) selection of the mRNA population by running the extracted RNA through an oligo(dT) cellulose column and (4) size fractionation of the isolated mRNA. At all stages, great care is required to ensure that the preparation does not come into contact with active ribonuclease enzymes which can destroy the RNA. Because the goal of the cDNA cloning procedure is to obtain "full length" cDNA clones that contain the entire coding sequence of the gene, it is extremely important to Use procedures that maintain the integrity of the mRNA. Ribonuclease (RNAse) enzymes are very stable and so even a very small amount of the active enzyme in an mRNA preparation will cause problems. RNAse is present on virtually all surfaces, including human skin, and is thus very easily introduced into the RNA preparation. To avoid contamination problems, all solutions, glassware and plasticware must be specially treated. The cells from which the mRNA is to be isolated are disrupted in solutions which are extremely harsh and contain components which immediately inactivate the omnipresent ribonuclease enzymes; all subsequent solutions used in RNA preparation are treated with diethylpyrocarbonate (DEPC) , a suspected carcinogen) which inactivates RNAse. Often a laboratory will set aside particular equipment and work space that is designated to be "ribonuclease free". The potential for RNA degradation starts at the first step of breaking open the cells (the cells themselves contain ribonucleases which, upon lysis of the cells, come into contact with the RNA) , and continue throughout the procedure.
Total RNA extracted from a cell is made up of messenger RNA (mRNA) , transfer RNA (tRNA) and ribosomal RNA (rRNA) . The mRNA typically makes up only 1-3% of the total cellular RNA (approximately 1 x I0"1g mRNA per eukaryotic cell, Sargent, T.D., Methods Enzymol . 152:423- 432 (1987)). Most cDNA synthesis reactions rely on the presence of the poly(A) tail present only in mature mRNA transcripts. The mature RNA transcripts are selectively extracted from the bulk of the cellular RNA, usually by affinity chromatography. This is an essential step for successful in vitro cDNA synthesis; failure to enrich for the mature mRNA frequently results in a low yield of poor quality cDNA.
As a final stage prior to cDNA synthesis, the mRNA preparation may be size selected. This is usually performed to remove the smaller size molecules (usually degraded forms of larger mRNAs) which would otherwise interfere with the cDNA cloning procedure. Size selection may also be performed to enrich for an mRNA species of known size. Size selection may be performed by electrophoresis through agarose gels, by column chromatography, or by sedimentation through sucrose gradients. These techniques result in lower yields, and may require the presence of methylmercuric hydroxide to disrupt secondary intramolecular structure. Methylmercuric hydroxide is extremely toxic and volatile, and requires great care in handling. Safer alternatives (such as the use of gels containing glyoxal/dimethyl sulfoxide or formaldehyde) are available, but these techniques also involve dangerous chemicals and have associated disadvantages.
Additional disadvantages in the present state of the art arise from the necessity of extracting mRNA from cells prior to cDNA synthesis. For example, cDNA cloning is often used to assess which genes are expressed in a cell under particular conditions or at a particular stage in the development of the organism. The time and conditions required to extract the mRNA may themselves produce alterations in the gene expression pattern of the cell. Furthermore, mRNA molecules which are present in very low abundance (estimated at 20 copies per cell) or which are unstable may be lost during the RNA isolation procedure. There is currently a lower limit on the number of cells necessary to produce a cDNA library due to the inherent losses incurred in mRNA isolation procedures. This invention addresses this problem by completely circumventing the initial mRNA isolation requirement.
Following extraction and purification of the mRNA, cDNA synthesis is performed in vitro. All methodologies presently used for cDNA synthesis follow mRNA extraction and purification, or are performed in situ or under in vitro conditions. These methodologies are reviewed in detail in Kimmel and Berger (1987); Okayama, H. , et al . , (1987) ; Van Gelder, et al . , Proc. Natl . Acad. Sci . USA 87:1663-1667 (1990); and Embleton, M.J., et al . , Nucleic Acids Res. 20:3831-3837 (1992)). All of the presently available techniques utilize RNA-dependent DNA polymerase enzymes (more commonly termed reverse transcriptase enzymes) to synthesize the first strand of the cDNA from the mRNA template.
Reverse transcriptase enzymes cannot initiate nucleic acid synthesis de novo. Rather, these enzymes add the first nucleotide of the nascent cDNA strand to the hydroxyl group of a molecule which is bound to the RNA template molecule. This bound molecule to which the enzyme adds the first nucleotide is called a primer and appears to be absolutely required for reverse transcriptase activity - regardless of the source of the enzyme. However, there appear to be major differences in the requirements for priming the initiation of cDNA synthesis under in vitro and in vivo conditions. Fulfilling the primer requirement for retroviral enzymes under in vitro (outside the cellular environment) or in situ cDNA synthesis conditions is elementary, and a short segment of deoxyribonucleic acid (DNA) , termed an oligonucleotide primer, is most commonly used. Frequently this primer is a polymer of deoxyribothymidylic acid (oligo(dT)). In all cDNA protocols currently available this DNA primer is designed simply to anneal to the RNA template molecule, and is not capable of forming a specific structure which contributes to the binding of the reverse transcriptase enzyme to form a primer-enzyme complex (Kimmel and Berger (1987) ; Okayama, H. , et al . , (1987); Van Gelder, et al . , Proc. Natl . Acad. Sci . USA 87:1663-1667 (1990); and Embleton, M.J., et al., Nucleic Acids Res. 20:3831-3837 (1992)). Alternatively, in vitro cDNA synthesis may utilize an oligonucleotide primer that is complementary to other sequences within the RNA molecule; however, because of the extensive stretch of complementary nucleotides necessary for annealing to occur, such a primer will be "sequence specific" for the mRNA molecule to which it is designed to anneal. Synthesis of such a sequence specific primer requires prior knowledge of the nucleotide sequence of part of the mRNA. The primer requirements of reverse transcriptase enzymes are discussed in Chapter 5 of Sambrook, et al . (1989).
In retroviruses, the reverse transcriptase enzymes are already complexed with a structure-specific cellular tRNA molecule which the virus scavenged from a host cell during the previous infection. Transfer RNA molecules are short (70 to 80 nucleotides long) RNA molecules which are folded into complex three-dimensional structures; their usual cellular role is in the translation process (Darnell et al . (1989), chapter 4). This reverse transcriptase enzyme complex is, in turn, bound to the site of cDNA synthesis initiation on the viral RNA template molecule, and serves as the primer-reverse transcription enzyme complex.
After the virus enters a newly infected cell the reverse transcriptase-tRNA complex acts as primer for an in vivo (in the cell) reverse transcriptase reaction, using the viral RNA molecule, to which this complex is already bound, as the RNA template.
The initial cDNA product made from this in vivo reaction is a short cDNA segment termed the "minus-strand strong-stop". This cDNA then serves as a primer molecule in an intramolecular or intermolecular priming event which allows resumption and completion of cDNA synthesis on the retroviral RNA template. The cDNA molecule is then converted to double-stranded DNA by the same reverse transcriptase enzyme complex, and integrated into a chromosome of the infected cell.
Just as in the retrovirus lifecycle, the initial product of the in vivo cDNA synthesis reaction in hepadnaviral replication is the formation of a "minus- strand DNA" product, and, again, similar to the strategy employed by retroviruses , this initial cDNA product acts to prime at a second polynucleotide site to recommence cDNA synthesis.
It is important to note that the reverse transcriptase-tRNA primer complex necessary for retroviral replication will not act as a primer for cDNA synthesis from most cellular mRNA templates. The cognate tRNA species (2 of about 40 tRNA molecules present in the cell) which have affinity for the reverse transcriptase enzyme, are complementary to, and therefore will only prime from sequences present on specific RNA template molecules. It is interesting to note, however, that there are virus-like 3OS (VL30) elements present in mouse cells which have regions of strong homology to retroviruses. These elements have properties of defective type C viruses, are reverse transcribed and are packaged into retroviral virions (Howk, R.S., et al . , J. Virol . 25:115-123 (1978); Besmer, P.U. , et al . , J. Virol . 29:1168-1176 (1979); Sherwin, S.A., et al . , J. Virol . 26:257-264 (1978); Scolnick, E.M. , et al . , J. Virol . 29:964-972 (1979J ; Scadden, D.T. , et al . , J. Virol . 64:424-427 (1990); Rodland, K.D., , et al . , Mol . Cell . Biol . 7:2296-2298 (1987); Hatzoglou, M. , et al . , Human Gene Therapy 1:385-397 (1990)). Similarly, the hepadnaviruses reverse transcriptase enzymes appear to require specific structural target elements within the hepadnaviral RNA template molecule in order to bind and initiate cDNA synthesis (Wang and Seeger, J. Virol . 67:6507-6512 (1993)). The product of the initial reverse transcriptase reaction in vitro is a single-stranded complementary DNA copy of the mRNA molecule. This reaction is often referred to as "first strand cDNA synthesis." Thereafter, various techniques are used to generate the second strand of the cDNA. The resultant double-stranded DNA (dsDNA) molecules are then modified at the ends, and inserted into a "vector" which allows growth, selection, and amplification of each copy. Most commonly used techniques (eg. Okayama and Berg, Molecular and Cellular Biology 2:161-170 (1982)) may be summarized as follows: Following extraction and purification of the mRNA and in vitro reverse transcription of the mRNA to produce single-stranded cDNA molecules, the mRNA template is eliminated to allow synthesis of the second strand of DNA and thereby form a double-stranded cDNA molecule; specific DNA linkers are then attached to the blunted end of the double-stranded cDNA,and the cDNA is ligated into a suitable cloning vector.
In all presently used techniques, the reverse transcriptase-catalyzed step of making a cDNA copy of the mRNA is always performed under in vitro conditions. The quality of the cDNA synthesis (that is, the ability to generate both accurate and full-length complementary DNA) depends upon the fidelity and the processivity of the enzyme chosen, and the conditions under which the reaction is performed. Clearly less than full-length cDNA is not acceptable, and a high error rate will compromise the utility of the cDNA produced. Reverse transcriptase enzymes, unlike their cellular DNA polymerase counterparts, lack enzymatic 3' -» 5' exonuclease (proofreading) activity so fidelity depends on base discrimination during polymerization. The use of the reverse transcriptase in vitro, rather than under the in vivo conditions which the enzyme has evolved to function, appears to adversely affect both the fidelity and processivity of the enzyme. The in vitro fidelity of MuLV reverse transcriptase has been estimated to be 10"4 (i.e. one wrong nucleotide per 10,000 bases or 10 errors per lOOkb) , and recent studies have determined that the in vivo fidelity is approximately 2xl0"5 (1 error for every 50,000 bases copied, 2 errors per lOOkb; Mont et al., J. Virol . 66:3683-3689 (1992)). In addition, it is difficult to obtain full length first strand synthesis in an artificial environment whereas the processivity of the enzyme in the in vivo cDNA synthesis reactions is excellent; with cDNA incorporation extending well past the lOkb range (see included data) . While conditions have been developed to optimize the performance of reverse transcriptase enzymes in vitro, these conditions do lead to a certain frequency of errors, and premature termination of first strand cDNA synthesis. It is clear that the in vitro conditions do not reflect the optimal conditions for the enzyme.
In addition, none of the presently known techniques for cDNA synthesis facilitate the priming and initiation of cDNA synthesis by providing a structure- and reverse transcriptase-specific polynucleotide target molecule. Nor do the present techniques for cDNA synthesis provide a mechanism whereby inclusion of genetic elements is possible using a polynucleotide template primer molecule whose cDNA product acts as a primer molecule at a second polynucleotide site where cDNA synthesis recommences and completes the linkage of the complementary DNA of the initial, and any subsequent polynucleotide molecules. Thus, present techniques for cDNA synthesis are limited by either the requirement that the mRNA be extracted, purified, or withdrawn from cells, or the performance of the reverse transcriptase enzyme under in vitro conditions. These factors limit: the ease of cDNA synthesis; the efficiency of cDNA synthesis (therefore requiring a larger number of cells to construct a representative cDNA library) ; the size of cDNA molecules that can be produced (thereby the genes that are readily clonable by this technique) ; the accuracy of cDNA synthesis in determining which genes are expressed under particular conditions; and the fidelity of the cDNA produced.
It is an object of the present invention to provide a technique of cDNA synthesis that does not require isolation, withdrawal or removal of polynucleotide template molecules from cells.
It is a further object of the present invention to provide a technique of cDNA synthesis that does not require in vitro activity of reverse transcriptase. It is also an object of the present invention to provide for cDNA synthesis wherein the efficiency of the technique, the fidelity of the cDNA produced and the size of cDNA that the technique is capable of producing are superior to all presently used techniques.
It is also an object of the present invention to increase the efficiency of priming cDNA synthesis utilizing a first polynucleotide template primer molecule the product of which acts as a primer for resumption of cDNA synthesis at a second polynucleotide site.
It is also an object of the present invention to provide a means of encoding a variety of genetic elements into the final cDNA products by utilizing a first polynucleotide template primer molecule the product of which acts as a primer for resumption of cDNA synthesis at a second polynucleotide site.
It is an additional object of the present invention to provide a means of determining the in vivo cell expression pattern for a eukaryotic cell. It is an additional object of the present invention to provide a means of producing an infectious replication-defective recombinant virus which initiates in vivo cDNA synthesis upon infection of viable cells.
Disclosure of the Invention The present invention relates to methods and compositions for the synthesis of complementary DNA copies of polynucleotide templates.
In accordance with the present invention, a method for synthesizing a complementary DNA copy of a plurality of polynucleotide molecules is provided.
The method comprises providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, the first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to the first region which enables a cDNA product of said molecule to anneal to at least one distinct polynucleotide template molecule. In addition, at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template primer molecule is provided. A mixture comprising the reverse transcriptase enzyme or enzyme complex and the polynucleotide template molecules is then formed and incubated under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to the polynucleotide template molecules.
A further aspect of the invention provides a method for producing a complementary DNA copy of a polynucleotide template molecule.
In this aspect, the method comprises providing at least one polynucleotide template molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, the first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to the first region which enables a cDNA product of the molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to the first region, which third region is sufficiently similar in sequence to the second region such that the complementary DNA product of the second region anneals to the third region so as to allow complementary DNA synthesis to recommence. In addition, at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template primer molecule is provided. A mixture comprising the reverse transcriptase enzyme or enzyme complex and the polynucleotide template molecule is then formed and incubated under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to the polynucleotide template molecule. An additional aspect of the invention provides a method for generating a primer-specific cell expression pattern in a viable cell.
This method comprises providing a reverse transcriptase-cognate primer transfer RNA molecule in which the 3'-region has been modified to selectively anneal to a subpopulation of cellular polynucleotide template molecules. In addition, at least one reverse transcriptase enzyme which initiates DNA synthesis from the primer molecule is provided. A mixture comprising the reverse transcriptase enzyme and the primer molecule is then formed in a viable cell and incubated under conditions which permit the synthesis of DNA molecules which comprises regions complementary to the cellular polynucleotide template molecules. Thereafter, the cDNA products thus produced are analyzed to form a pattern therefrom.
Alternatively such a method is accomplished by providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a subpopulation of cellular polynucleotide template molecules. This primer molecule is then utilized in place of the reverse transcriptase- cognate primer transfer RNA molecule in the method just described.
Also provided in accordance with the present invention are compositions useful in the practice of the present method. Such compositions include polynucleotide template primer molecules and recombinant viral particles which are capable of initiating in vivo cDNA synthesis upon introduction into a viable eukaryotic cell. Such recombinant viral particles comprise at least one polynucleotide template primer molecule which includes the functional sequence equivalents of the sequence elements required for viral packaging (e.g. the e region in hepadnaviruses; Ψ, DR, and Ψ+ regions in retroviruses) , a region which binds a reverse transcriptase enzyme or complex thereof, and a 5' sequence the cDNA product of which will serve to prime a resumption of cDNA synthesis at a second polynucleotide site. Such recombinant viral particles will find use in a mixture which provides all other required trans-acting elements (e.g. capsid, nucleocapsid proteins) which will result in the packaging of the polynucleotide template primer molecule into an infectious particle.
Further compositions provided in the invention include Genetic Elements (GE) template primer polynucleotide molecules which bind at least one reverse transcriptase enzyme or complex thereof and serve as a template molecule for the synthesis of a complementary DNA copy of the template primer molecule. The cDNA product of the 5'-region of this template primer molecule will then act to prime the resumption of cDNA synthesis at least one other site on a polynucleotide molecule. The cDNA synthesis reaction covalently links the cDNA products of at least one polynucleotide template. Compositions of the invention also include DNA molecules and recombinant DNA vectors encoding the polynucleotide molecules of the invention, and kits containing such compositions. Brief Description of the Drawings
Figure 1 depicts a simplified diagram of the eukaryotic RNA transcription process, illustrating removal of the non-coding intron sequences during mRNA processing;
Figure 2 depicts the cloning of gene cassettes in the present invention, in which
Figure 2A depicts a schematic diagram of certain techniques involved in cloning of the tRNAPro gene cassettes; and
Figure 2B depicts the structure and sequence of a tRNA molecule corresponding to the "wild type" murine tRNAlJ™ utilized by Moloney murine leukemia virus, in which the solid line defines the 3' nucleotide sequence which anneals to the virion RNA template and primes the retroviral reverse transcriptase reaction; Figure 3 depicts the relationships involved in reverse transcriptase, tRNA primer, and RNA template interactions necessary for cDNA synthesis, in which Figure 3A depicts retroviral cDNA synthesis sequences for both the 3'-region of the cognate tRNA| 0utilized by the Moloney murine leukemia virus, and the complementary Primer Binding Site on the retroviral RNA segment (straight solid line) , to which the tRNA binds, and the initial stages of retroviral replication (the dashed line represents cDNA synthesis) ; and
Figure 3B depicts in vivo cDNA synthesis from a polyadenylated messenger RNA, in which sequences are shown for both the 3'-region of the
Figure imgf000021_0001
which has been modified and synthesized in vitro, and the mRNA polyadenylated 3'-segment to which the tRNA binds, as well as the initial stages of in vivo cDNA synthesis (the dashed line) ; Figure 4 depicts the steps involved in the amplification-mutagenesis of the tRNA-encoding promoter- tRNA cassette, wherein the first and second primers are represented by dashed lines with arrows delineating the 5' to 3' orientation, the second primer is represented by a bent dashed line to illustrate the inability of 5' bases to anneal to uncomplimentary bases in the initial template DNA molecule, and the bottom schematic represents the amplified and modified transcription cassette in which the 3'-most bases have been changed to the prescribed sequence encoded in the second primer, and the 3'-end of the cassette is truncated at the end of this second primer sequence;
Figure 5 is a photographic reproduction of an ethidium bromide-stained 1% agarose/TAE gel of in vivo cDNA products from insect Sf9 cells, in which lanes 1 and 10 represent a lkb ladder (BRL) , lanes 2 and 6 represent control ([α-32P]dCTP alone), lanes 3 and 7 represent control ([α-3P]dCTP + reverse transcriptase), lanes 4 and 8 represent control ([α-3P]dCTP + reverse transcriptase + tRNAMt) , lanes 5, 9, 11 and 12 represent experimental with modified tRNA primer ([c.-32P]dCTP + reverse transcriptase + tRNAp^) . In this example, the samples in lanes 11 and 12 are not treated with RNase A, the samples in lanes 2- 5, and 11 are electroporations in 0.4cm cuvettes, and the samples in lanes 6-9, and 12 are electroporations in 0.2cm cuvettes;
Figure 6 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 5; Figure 7 is a photographic reproduction of an ethidium bromide stained 1% agarose/TAE gel of in vivo cDNA products from Hamster (CHO) cells, in which lanes 1 and 10 represent a lkb ladder (BRL) , lanes 2 and 6 represent control ([α_3P]dCTP alone), lanes 3 and 7 represent control ([α-3P]dCTP + reverse transcriptase). lanes 4 and 8 represent control ([α_32P]dCTP + reverse transcriptase + tRNAyt) , lanes 5 and 9 represent experimental with modified tRNA primer ([α~32P]dCTP + reverse transcriptase + tRNApol^) ; Figure 8 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 6;
Figure 9 is a photographic reproduction of an ethidium bromide stained 1% agarose/TAE gel of in vivo cDNA products from Hamster (CHO) cells, in which lanes 1 and 8 represent a lkb ladder (BRL) , lanes 2 and 3 represent control ([α-32P]dCTP + reverse transcriptase + tRNAut) , lanes 4 and 5 represent experimental with modified tRNA primer ([α_3P]dCTP + reverse transcriptase + tRNApolyU) , and lanes 6 and 7 represent control ([α"32P]dCTP + reverse transcriptase + oligo(dT) (5//g) . In this example, the samples in lanes 3, 5, and 7 are treated with SI nuclease;
Figure 10 is a photographic reproduction of an autoradiograph of the gel image depicted in Figure 9; Figure 11 depicts a simplified diagram of retroviral proviral synthesis. The dashed line represents the viral RNA genomic segment with the tRNA primer (looped structure) and reverse transcriptase (RT) annealed to the complementary sequence of the retrovirus-specific Primer Binding Site (PBS) . The "R" regions are repeated sequences present near each end of the genomic RNA segment. The heavy solid lines represent cDNA synthesis directed by the RNA template sequences;
Figure 12 depicts a simplified diagram of a representative embodiment of in vivo cDNA synthesis in the presence of Vector Control Elements (VCE) RNA template. The dashed line represents an RNA molecule. PolyA tract refers to polyadenylic acid — one choice for the 5'-terminal sequence of the RNA template molecule. "Neo" refers to an RNA sequence which codes for a region which confers biological resistance to the antibiotic G418 in eukaryotes (neomycin in prokaryotes) . "Ori" is a commonly accepted abbreviation for the origin of replication of a polynucleotide molecule in a biological host. The arrow which encompasses the word "promoter" indicates one orientation of a promoter element operatively linked to any polynucleotide segment which encodes a biological molecule; and
Figure 13 depicts aspects involved in the generation of a primer-specific cell expression pattern in viable cells, in which ,
Figure 13A provides a diagrammatic representation of
RNA transcripts and their associated sizes present in cell populations A and B; and Figure 13B depicts one possible pattern of cDNA products resulting from the in vivo cDNA synthesis employing a specific template primer or modified cognate tRNA primer molecule.
Detailed Description of the Invention The present invention relates to methods and compositions for the synthesis of complementary DNA (cDNA) . This invention provides, for the first time, a method by which cDNA synthesis is made possible utilizing a structure-specific analog to the natural target site of the reverse transcriptase enzyme or reverse transcriptase enzyme complex. In this method, suitable template, or template primer molecules which have a first site which binds to a reverse transcriptase enzyme or reverse transcriptase enzyme complex is introduced into a mixture in the presence of a reverse transcriptase enzyme, or reverse transcriptase enzyme complex, and the mixture is incubated under conditions such that a DNA molecule complementary to at least one polynucleotide template molecule(s) is produced. In addition, production of a pattern of cDNA products which reflects the quantity and molecular size of cellular transcripts present in the viable cell at a specific time, or in response to a specific stimulus is now made possible by the present invention. This in vivo cell- and primer-specific "bar-code" or "transcript fingerprint" pattern will prove very useful for many purposes including research, clinical diagnosis and forensics. This technology would provide to genetics analysis the "expressed gene" equivalent to restriction fragment length polymorphism (RFLP) analysis of total genomic DNA.
In the present invention, the requirements for priming cDNA synthesis in an in vivo environment appear to be more stringent than in vitro, and oligo(dT) primer molecules, and other DNA primers appear not to function under in vivo cDNA synthesis conditions (see Example section) . To date, all known reverse transcriptase enzymes functioning under in vivo conditions are directed to the site of cDNA synthesis initiation by a structure- specific polynucleotide molecule. Indeed, even the reverse transcriptase enzymes from hepadnaviruses, which utilize a hydroxyl group side chain from a tyrosine residue derived from the reverse transcriptase enzyme itself to prime cDNA synthesis (Wang and Seeger, Cell 71:663-670 (1992); Wang and Seeger, J. Virol . 67:6507- 6512 (1993)), are targeted to the site of hepadnaviral cDNA synthesis initiation by the structural sequence elements of the template specific binding site, epsilon (e) (Weber, M. et al . , J. Virol . 68:2994-2999 (1994)). Therefore, the first step involved in initiation of all in vivo cDNA synthesis reactions appears to be the directed binding of the reverse transcriptase enzyme to a structure-specific polynucleotide target. In retroviruses, this role of structure-specific polynucleotide molecule is satisfied by cellular molecule(s) referred to as the reverse transcriptase cognate transfer RNA (tRNA) molecule(s) . They are designated "cognate" for two reasons: (1) the reverse transcriptase enzyme produced by that specific retrovirus recognizes and binds to the specific structure of a very limited subset of cellular tRNA molecules, and (2) the 3'-region of the cognate tRNA molecule, in turn, binds to a complementary sequence at the site of cDNA initiation (Primer Binding Site) which is specific for the retrovirus, and is encoded in the retrovirus RNA genomic template molecule.
In hepadnaviruses, the role of structure-specific polynucleotide molecule is fulfilled by the hepadnavirus pregenomic RNA template specific binding sites, epsilon (e) , which appear to serve as the hepadnaviruses analog to the retrovirus cognate tRNA molecules.
In certain embodiments of the invention, the polynucleotide template, or template primer molecule is
RNA, and in many embodiments, the polynucleotide template or template primer molecule is a modified retroviral or hepadnavirus template RNA molecule. In addition, the invention provides, for the first time, a means by which all of the genetic elements necessary for biological selection, replication and amplification, integration and expression of an heterologous genetic element can be covalently included as a contiguous cDNA product in an in vivo process; therefore many of the crucial steps of molecular cloning are performed by the viable cell in the in vivo intracellular environment. This genetic control information is encoded in a Genetic Elements (GE) polynucleotide template, or template primer molecule. In certain embodiments of the invention, this polynucleotide template, or template primer molecule is transcribed within the viable cell. In many embodiments, this GE polynucleotide template, or template primer molecule is introduced into a viable target cell in the presence of a suitable reverse transcriptase enzyme, or reverse transcriptase enzyme complex.
The GE polynucleotide template, or template primer molecules, when used in combination with a retroviral reverse transcriptase enzyme and a modified cognate tRNA molecule, may include sequence elements which will form structures similar to those of the U5-leader and U5-IR stems in the retroviral genomic RNA template. These sequences have been shown to be important in viral replication (Cobrink, D. , et al . , J. Virol . 62:3622-3630 (1988)), and appear to interact with the TΨC loop of the tRNA primer to significantly improve the efficiency in priming cDNA synthesis (Cobrink, D. , et al . , J. Virol . 65:3864-3872 (1991); Murphy and Goff, J. Virol . 63:319- 327 (1989); Aiyar, A., et al., J. Virol . 66:2464-2472 (1992)). Therefore, a modified tRNA primer molecule may interact in an energetically more favorable manner with RNA template sequences which reproduce an analogous RNA structure. In addition, inclusion of nucleocapsid protein (or other proteins) may facilitate the formation of an initiation complex composed of the RNA template, tRNA primer and reverse transcriptase enzyme (Meric and Goff, J. Virol . 63:1558-1568 (1989); Khan and Giedroc, J. Biol . Chem. 267:6689-6695 (1992)), thus improving the efficiency of cDNA synthesis.
Alternatively, when the GE polynucleotide template, or template primer molecules are used in combination with a hepadnavirus reverse transcriptase enzyme they may include sequence elements which will form structures similar to those of the specific binding site, epsilon (e) pregenomic RNA template. These sequence elements have been shown to be critical for both the initiation of cDNA synthesis, and packaging of the hepadnavirus genome. Inclusion of sequences in the 5'-end of the initial polynucleotide template molecule which are represented in the 3'-end of the same template molecule will facilitate intramolecular annealing of the initial minus-strand strong-stop cDNA product with these 3'-sequences. This will result in intramolecular priming at a second polynucleotide site, where resumption of cDNA synthesis will occur.
Unless otherwise defined, all technical and scientific terms will be used in accordance with the common understanding of persons ordinarily skilled in the art to which the present invention is related. As used herein, the following terms shall have the assigned meanings unless a contrary definition is clearly indicated from the context in which the term is used.
The term template indicates a nucleotide sequence from which a complementary sequence is produced. The term template primer indicates a polynucleotide sequence from which cDNA synthesis is initiated and extended, and the cDNA product of said template primer molecule serves to prime the resumption of cDNA synthesis at a second polynucleotide site. The terms Genetic Elements (GE) and Vector Control Elements (VCE) template primer molecule are used interchangeably and indicate a template primer molecule which may include encoded genetic regions for functions including, but not limited to: amplification, selection, replication, insertion, segregation, integration, excision, stabilization, purification, expression, detection, localization, processing, or packaging in prokaryotic or eukaryotic cells. The term analog is used to indicate any sequence- specific representative of a naturally-occurring nucleotide.
The term reverse transcriptase enzyme is taken to mean any polymerase which can catalyze the addition of a deoxynucleotide or analog thereof to a primer annealed to an RNA template.
The term polynucleotide includes homo- and hetero- polymers of deoxyribonucleic acids, ribonucleic acids and analogs thereof.
The term modified reverse transcriptase-cognate primer transfer RNA molecule refers to any tRNA molecule, whether produced chemically, by means of an artificial biological system, or purified from a biological source, which is modified so as to prime the activity of a specific reverse transcriptase enzyme.
The term Primer Binding Site ("PBS") refers to the specific nucleotide sequence in an RNA template molecule to which the naturally-occurring cognate primer of a specific reverse transcriptase enzyme anneals to initiate cDNA synthesis. Therefore, any nucleotide additions, deletions or other modifications in the naturally- occurring cognate primer which result in variations in the RNA template sequence to which the primer anneals constitute binding at a site other than the Primer Binding Site.
The term R region sequence refers to the natural repeated sequences found at both the 3'- and 5'-ends of retroviral genomic RNA. The term expression cassette refers to a DNA construct which includes all sequences necessary for the expression of the coded product. Accordingly, an expression cassette will include DNA encoding at least a promoter region, the sequence of interest and a transcription termination region. The terms vector and plasmid are interchangeably used to include any means which permits DNA to be replicated and selected in a particular system.
The term operative linkage refers to nucleotides which are joined in a manner which preserves the functional relationship between the sequences on each side of the linkage. For example, a promoter operatively linked to a DNA sequence will be upstream both with respect to the direction of transcription and with respect to the transcription initiation site and inserted in a manner such that transcription elongation proceeds through the DNA sequence.
Many embodiments of the present method of cDNA synthesis do not require the isolation of mRNA from cells. Such cDNA synthesis eliminates the problems associated with conventional in vitro techniques where: 1) mRNA isolation is required; 2) the cellular components are withdrawn into an artificial environment; or, 3) the cells are killed and subjected to harsh conditions. Therefore, the mRNA templates are more likely to be intact and full length cDNA clones can more reliably be obtained. Furthermore, in vivo cDNA synthesis does not require the in vitro activity of reverse transcriptase, but rather permits the reverse transcription step to be performed in vivo (i.e. within the cellular environment) , such that the efficiency, fidelity and processivity of the reverse transcriptase enzyme is optimized. Thus, longer cDNA clones, with few nucleotide sequence errors may be produced by this method. In addition, it is easier to perform, requires a considerably shorter period of time, and the procedure can potentially provide cDNA product starting with a smaller number of initial cells.
As described in the background of the invention section above, conventional cDNA synthesis requires isolation and purification of mRNA from cells, or the withdrawal of the reaction components into an artificial environment, followed by an in vitro cDNA synthesis step. In the in vitro cDNA synthesis step, the requirement for a primer for the reverse transcriptase enzyme is most commonly met by supplying an oligo(dT) primer molecule that anneals to the poly(A) tail of mRNA molecules, or an oligonucleotide molecule that is complementary to a known portion of target mRNA sequence. However, neither of these types of oligonucleotide primers appear to be effective in vivo (see Examples below) . Although specific evidence is lacking to confirm why simple polynucleotide oligomers of complementary sequence which successfully prime in vitro cDNA synthesis reactions fail to prime in vivo cDNA synthesis reactions, there are at least two possible explanations to ascribe for the failure: 1) the retroviral reverse transcriptase enzymes are known to recognize and associate with cognate tRNA molecules which have a specific sequence and, therefore, three dimensional structure. This has been studied in detail with both natural and synthetic tRNA molecules by means of gel mobility shift experiments with the reverse transcriptase enzyme from HIV (Barat, C. , et al . , EMBO J. 8:3279-3285 (1989); Barat, C, et al . , Nucleic Acids Res. 19:751-757 (1991); Weiss, S., et al . , Gene 111:183-197 (1992)). Simple oligomer primers do not contain this structural requirement, and so, the retroviral reverse transcriptase enzymes may not recognize and associate with the primer under in vivo conditions, even if the polynucleotide primer is able to anneal to the mRNA template. Likewise, hepadnaviral reverse transcriptase enzymes recognize and bind to a specific structural region, epsilon (e) , present on the pregenomic RNA template molecule (Weber, M. et al., J. Virol . 68:2994- 2999 (1994)). Mutations in the e region interfere with the formation of minus strand DNA (page 6508, Wang & Seeger, J. Virol . 67:6507-6512 (1993)), which serves as an obligate primer for plus-strand synthesis at DR1. 2) Alternatively, the polynucleotide primer which anneals to the template under in vitro conditions, simply may be unable to anneal efficiently to an mRNA template in vivo . In the natural life-cycle of retroviruses, viral reverse transcriptase enzymes utilize host tRNA molecules as primers in order to synthesize a DNA copy of the single-stranded retroviral RNA genome. There may be more than 40 different types of tRNAs in animal cells. Each infective retrovirus particle contains two copies of a single-stranded viral RNA chromosome each of which is associated with a specific host tRNA molecule which anneals to a particular region of the retroviral RNA termed the Primer Binding Site. To initiate the reverse transcription process, a sequence of bases at the 3'-end of the tRNA anneals to the Primer Binding Site of the retroviral RNA. The reverse transcriptase enzyme (which is already associated with this complex) then utilizes this tRNA as a primer molecule, adding the first nucleotide of the nascent DNA molecule to the 3'-hydroxy terminus of the tRNA.
The specificity of priming retroviral reverse transcription is determined by the base pair sequence at the 3'-end of the tRNA molecule which anneals to the retroviral genome. Each retrovirus utilizes a tRNA primer capable of annealing to the specific Primer Binding Site sequence present in the retrovirus genome. For example, the human immunodeficiency virus (HIV) genome utilizes tRNA^ as a primer. To initiate synthesis of a DNA copy of the HIV virus, eighteen nucleotides at the 3'-end of the tRNAL 3 ys unfold and base pair with the HIV Primer Binding Site (Weiss et al . , RNA Tumor Viruses. Cold Spring Harbor ( 982) > Goff, J. Acquired Immune Deficiency Syndrome 3:817-831 (1990)). Thus, the eighteen nucleotides at the 3'-terminal of the tRNA^ys are complementary to the HIV Primer Binding Site sequence. In addition to this annealing of the tRNA primer to the viral RNA template, portion(s) of the same tRNA molecule will be recognized by the viral reverse transcriptase enzyme so that a trimolecular complex is ultimately formed (tRNA primer-reverse transcriptase-RNA template) .
Therefore, for all in vivo cDNA synthesis reactions, a polynucleotide template or template primer molecule should fulfill two criteria. Firstly, the molecule should be able to bind a reverse transcriptase enzyme or reverse transcriptase enzyme complex; secondly, the bound enzyme or enzyme complex must initiate cDNA synthesis from the region of that site and produce an initial cDNA molecule which is complementary to the polynucleotide template. The simple oligonucleotide primers presently used with retroviral reverse transcriptase enzymes to initiate in vitro cDNA synthesis do not function in vivo (see data, included) . Furthermore, while specific tRNA molecules are able to function as primers for the in vivo action of reverse transcriptase on the retroviral genome, these tRNA primers anneal specifically to the retroviral Primer Binding Site and are not designed to anneal to sequences present in all mature cellular mRNA molecules. Similarly, reverse transcriptase enzymes from hepadnaviruses will recognize and bind to the specific binding site (e) present in the hepadnaviral pregenomic RNA template molecule, but will not bind or initiate cDNA synthesis from regions present on all mature cellular mRNA molecules.
The present invention provides a method of in vivo cDNA synthesis by providing template primer molecules whose cDNA products will anneal to all polyadenylated cellular mRNA molecules, and are utilized by the reverse transcriptase enzyme such that a cDNA copy of the mRNA molecule to which the primer molecule anneals is produced. This invention is analogous to retroviral and hepadnaviruses replication processes (actually in vivo cDNA synthesis reactions) substituting modified retroviral or hepadnaviral template molecules, or template primer molecules, for the viral template molecules.
In an embodiment of the present invention, the template primer molecule is a modified retroviral template molecule wherein the 5'-region of the molecule is replaced with a polyadenylic acid sequence such that the cDNA product of the template anneals to the 3'- poly(A) tail that is present on mature cellular mRNA molecules. In another embodiment of the present invention, the template primer molecule is a modified hepadnaviral template molecule wherein the 5'-region of the molecule is replaced with a polyadenylic acid sequence such that the cDNA product of the template anneals to the 3'- poly(A) tail that is present on mature cellular mRNA molecules.
Such preferred template primer molecules may be used to synthesize in vivo cDNA copies of all mature mRNA molecules contained within a viable cell. Because the product of this template primer will anneal to all mature mRNA molecules, the template primer may be used to produce a cDNA library, that is a collection of cDNA molecules that represents all of the structural genes being expressed in the cell at that given point in time. In one embodiment of the invention the cDNA synthesis reaction takes place in viable cells under in vivo conditions, without subjecting the cells to the stress and perturbation of mRNA extraction, isolation, or withdrawing the cellular contents into an artificial in vitro environment. Therefore the cDNA synthesis products more accurately reflect the snapshot of mRNA populations within the cell at the time that the cDNA synthesis was initiated, and the products are synthesized in an in vivo environment. In addition, there are no artificial amplification steps necessary to produce a library from a small number of initial cells, and libraries which contain rare members may be more readily produced from limited quantities of specialized cells (e.g. pluripotent hematopoietic stem cells) . In another embodiment of the present invention, an initial specific polynucleotide template primer molecule is utilized to both increase the annealing and priming potential of the nascent cDNA transcript, and to provide a means to incorporate additional encoded genetic elements, or vector control elements into the final in vivo cDNA products.
In another embodiment of the present invention, the 5'-region of the template primer molecule is modified so that it is complementary to part of a specific polynucleotide sequence. This requires that either the nucleotide sequence of a desired polynucleotide template molecule is already known, or that one wishes to produce cDNA from a population of template molecules which contains that complementary sequence. Synthesis of specific cDNA molecules may be useful in many different ways, including, but not limited to the following: A sequence-specific template primer may be utilized to clone cDNAs with known sequence, to clone from genes which represent a specific gene family (all of which share a common sequence) , or to determine ratios of splice variants on two separate unrelated transcripts.
In addition, a template primer molecule can be used for intracellular mutagenesis or cDNA coupling reactions; a template primer molecule which encodes desired sequence elements 3' to a 5' sequence, the cDNA product of which will anneal to a distinct cellular transcript and prime the resumption of cDNA synthesis 5' to a region which one desires to mutate, will allow replacement of the cellular transcript sequences 3' to the site of template primer cDNA product annealing - with the concomitant loss of the cellular transcript information 3' to the site of this template primer annealing.
A template primer having a sequence which includes a number of polyadenylic acid residues located 3' to a 5'- terminal specific polynucleotide sequence will allow the cDNA product of the template primer molecule to reposition itself and act as a primer on a second polynucleotide template molecule (e.g. at the junctional region proximal to the polyadenylic acid stretch present on mature messenger RNAs) , such that a snapshot of members containing a complementary junctional sequence will be converted to cDNA product. Stringency in priming cDNA synthesis will roughly correspond to a statistical formula, l/4n, where n=number of specific bases added to the 5'-end of the polyadenylic acid region of the template primer (e.g. if 5 bases are sequence specific followed by a series of 30-50 adenylic acid residues, then 1/45=1/1024 messages might be expected to be primed by the cDNA product and converted to cDNA) . If a cell expresses 10,000 different messages, then approximately 10 products might be expected to be primed and converted to a cDNA product with such a template primer. It is important to note that there are other facts which will affect the resulting primer-and cell-specific pattern which is generated: RNA template size and frequency, sensitivity of cDNA product detection, choice of nucleotide analog incorporated, priming at regions in the mRNA template other than at the 3'-end, and other factors affecting the efficiency of cDNA synthesis are examples. Following extraction, fractionation, and detection this technology allows a primer-specific cell expression pattern to be developed. When this pattern is generated with the proper primer sequence, the pattern will be specific for the type or developmental stage of a cell or tissue. The pattern may be diagnostic for cellular perturbation (e.g. transformation or infection) , or change in response to an applied stimulus. Changes may be characterized solely by changes in the density of various cDNA product which make up the pattern (See Figure 13) .
This is a particularly useful and powerful diagnostic tool because the amount of tissue removed in a surgical biopsy is sufficient to enable a technician to develop a primer-selective cell expression pattern which may be used for diagnosis — without resort to amplification technologies which often result in the introduction of artifacts. This pattern technology will be a powerful tool in diagnosis once pattern members are identified. Alternatively, a template primer which is selective for an RNA template can be used for in vivo DNA sequencing reactions to determine the 5'-sequence of a desired RNA template, without the need to clone and isolate the template. In addition, where the expression of a particular mRNA species is a reliable indicator of a particular disease condition, in vivo cDNA synthesis may be a useful alternative to polymerase chain reaction (PCR) based diagnostic techniques, or could be utilized as a therapeutic agent in conjunction with such diagnostic techniques. Polymerase chain reaction (PCR) has been used for this purpose in the past and is, at best, unreliable and biased due to problems which occur during amplification (Gilliland, G. , et al. in: PCR Protocols: A Guide to Methods and Applications, Academic Press (1990)) .
A specific primer can then be used to initiate in vivo cDNA synthesis within the aberrant cell as a form of antisense therapy based upon the ability of the reaction to convert existing mRNA templates into a cDNA product which is no longer capable of being translated. In another example, an HIV-specific complementary primer could incorporate modified deoxynucleotide bases into a cDNA which could be used for detection, sequencing or sorting (Link, H, et al . , J. Med. Virol . 37:143-148 (1992); Prober, J.M. et al . (1989)), or the bases could be designed such that the incorporated nucleotide analogs could be made cytotoxic, so that only infected CD4 cells would be eliminated, leaving uninfected CD4 cells viable. Thus, the present invention includes template and template primer molecules that may be used for in vivo cDNA synthesis. More specifically and in preferred embodiments, these molecules are modified retroviral or hepadnaviral RNA template molecules.
One skilled in the art will recognize that the practice of this invention does not require that the template or template primer molecules used for cDNA synthesis be derived from a preexisting viral genomic template molecule. Thus, any polynucleotide that is capable of (a) binding a reverse transcriptase enzyme or enzyme complex and (b) acting as a template for a reverse transcriptase enzyme or enzyme complex such that initiation and synthesis of a DNA molecule complementary to that polynucleotide template molecule can occur, can be utilized in the cDNA synthesis method of the present invention. The suitability of a particular template or template primer molecule for use in cDNA synthesis may be assessed by performing cDNA synthesis as described below and assaying the products of that synthesis. The template (or template primer) and the reverse transcriptase enzyme (or reverse transcriptase enzyme complex) may either be present in the cell, or either one or both can be synthesized in or introduced into the cell. Thus, in one embodiment of the present invention, in vivo cDNA synthesis may be performed in a cell line that carries genes encoding a reverse transcriptase enzyme and the template (or template primer) molecule. The expression of these genes will result in the synthesis of the reverse transcriptase enzyme and the template (or template primer) in the cell. In other embodiments, the genes encoding the reverse transcriptase and the template (or template primer) molecules may be expressed under the control of inducible promoters such that the induction of the genes will lead to the expression of the reverse transcriptase and the template molecules and thereby initiate cDNA synthesis.
More commonly, the template (or template primer) molecules and the reverse transcriptase enzyme (or reverse transcriptase enzyme complex) will be introduced into the cell from an external source. A number of techniques have been established for the delivery of biological materials into viable cells. Many of these techniques have been described (Molecular Cloning; A Laboratory Manual Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY. (1989); Keown, W.A. et al . , Meth. Enzymol . 185:527-537 (1990)), and include, but are not limited to, electroporation (Neumann, E. et al . , EMBO J. 1:841-845 (1982); Electroporation and electrofusion in cell biology Neumann, E. et al . eds., Plenum Press, New York (1989); Kamdar, P. et al . , Nucleic Acids Res. 20:3526 (1992); Rols, M-P. et al . , Eur. J. Biochem. 206:115-121 (1992) ; Tsongalis, G.J., et al., Mutat. i?es. 244:257-263 (1990); Lambert, H. , et al . , Biochem. Cell Biol . 68:729-734 (1990); Yorifuji, T. , and H. Mikawa, Mutat. Res. 243:121-126 (1990); Winegar, R.A. , et al . , Mutat. Res. 225:49-53 (1989)); DEAE-dextran (Levesque, J.P., et al . , Biotechniques 11:313-4, 316-8 (1991); Ishikawa, Y., and C.J. Homey, Nucleic Acids Res. 20:4367 (1992)); cationic liposomes (Jarnagin, W.R. et al . , Nucleic Acids Res. 20:4205-4211 (1992)) or cationic lipids (Walker, C. et al . , Proc. Natl . Acad. Sci . USA 89:7915-7918 (1992) ; receptor-mediated delivery (Wagner, E. et al . , Proc. Natl . Acad. Sci . USA 89:6099-6103 (1992); microinjection (Martin, P. et al . , Dev. Biol . ,
117:574-580 (1986)); protoplast fusion; laser or particle bombardment; and viral vector delivery. One skilled in the art will recognize that each of these techniques has associated advantages and disadvantages and will be able to select the delivery technique most suitable for the cell type being used.
In certain embodiments of the present invention, techniques for delivery of the in vivo cDNA synthesis primers, templates and template primers into cells includes electroporation and liposomal transfection. These technique can be used, as described below, to introduce modified tRNA primers, reverse transcriptase enzymes, and modified deoxynucleoside triphosphates into a number of distinct cell types. When the reverse transcriptase enzyme is introduced into the cell from an external source, the choice of in vivo cDNA synthesis primers, templates and template primers will be determined by the type of the reverse transcriptase enzyme selected. There are two commonly used and commercially available types of retroviral reverse transcriptase: Avian Myeloblastosis Virus reverse transcriptase and Moloney Murine Leukemia Virus reverse transcriptase. These enzymes are available on a commercial basis from such vendors as: Stratagene, 11011 N. Torrey Pines Road, La Jolla, California 92037; Bethesda Research Laboratories, Inc., P.O. Box 6009, Gaithersberg, Maryland, 20877; New England Bio Labs, Inc., 32 Tozer Road, Beverly, Massachusetts, 01915; and Boehringer Mannheim Biochemicals, 9115 Hague Road, P.O. Box 50816, Indianapolis, Indiana, 46250. The various enzymes have both inherent parameters: fidelity (error rate) , processivity (ability to complete cDNA synthesis) , and structure (heterodimer vs monomer) ; and marketing parameters, such as availability and acceptance, which are factors which should be considered in choosing the enzyme. In addition, it may be important to use a reverse transcriptase enzyme derived from a retrovirus or retroelement which functions in the cell type and organism of the embodiment (e.g. use a reverse transcriptase enzyme from a human retrovirus to synthesize cDNA in human cells) .
For the purposes of the present invention, a DNA cassette containing the encoded modified tRNA sequence operatively linked to a promoter sequence was cloned into a bacterial vector. This allowed production of the modified tRNA primer in vitro, using an RNA polymerase which recognized the promoter sequence. The choice of promoter for use in instances where the in vivo cDNA synthesis primer is to be transcribed in vitro will be dictated by the polymerase enzyme to be used in the selected in vitro transcription system. In a preferred embodiment of the present invention, the modified tRNAproGGG primer was expressed under the control of a bacteriophage T7 promoter in an in vitro transcription system using the T7 RNA polymerase enzyme. The modified tRNA primer produced was isolated and introduced into cells along with the reverse transcriptase enzyme, a modified MoMLV template primer molecule and radiolabeled deoxynucleotide. Following introduction of these exogenous components, the cell was incubated for a sufficient period of time and under suitable conditions to allow in vivo cDNA synthesis to occur. Suitable conditions are generally those under which the cell type in question is usually grown. In preferred embodiments of the present invention, the target cell is incubated for 0.5-2 hours under normal culture conditions for that cell type following introduction of the primer and reverse transcriptase enzyme. As described in the examples listed below, the introduction of a radiolabeled dNTP (for example, [α-32P]dCTP) may be used to determine the efficacy of in vivo cDNA synthesis under particular conditions and with particular primers. After incubation of the cells, the cDNA produced in vivo was then extracted from the target cell utilizing the Hirt- extraction protocol (Hirt, B. , J. Mol . Biol . 26:365-369 (1967)). The extracted cDNA product appeared to be double-stranded nucleic acid which was successfully cloned using common cloning procedures. As alternative methodologies, the primer, templates and template primers and/or enzyme could be produced from genes introduced into cells; the enzyme could be expressed from a gene within the cell, and the primer, templates and template primers introduced into the cell; or the primer, templates and template primers could be expressed within the cell and the reverse transcriptase could be introduced from the outside. The inclusion of modified (radiolabeled) deoxynucleoside triphosphates in the reaction was used as a tool to follow the incorporation of deoxynucleotides and is not a requirement of the method; however, it should be noted that inclusion of modified deoxynucleotides provides both a convenient technique of following incorporation and synthesis (when α-labeled deoxynucleotides are used) , and provides a means of selecting or distinguishing the products of an in vivo cDNA incorporation reaction (using biotinylated or other deoxynucleotide analogs) . Modified deoxynucleotides and deoxynucleotide analogs have been used successfully in all types of polymerase reactions and are obvious techniques to one skilled in the art (Prober, J.M. et al . , Science 238:336-341 (1987); Klevan, L. and G. Gebeyehu, Meth. Enzymol . , 154:561-577 (1987); Chan, V. T-W. , et al . , Nucleic Acids Res. 13:8083-8091 (1985); Lo, Y-M. D. , et al . , Nucleic Acids Res. 16:8719 (1988)) . The products of these reactions can be used in various ways including, but not limited to: the construction of subtractive cDNA libraries; production of specific cytotoxic or light sensitive cDNA products; in vivo DNA sequencing; and cDNA probes for analytical, diagnostic or preparative use.
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
EXPERIMENTAL
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents) ;
M (Molar) ; mM (millimolar) ; μV (micromolar) ; N (Normal) ; mol (moles) ; mmol (millimoles) ; //mol (micromoles) ; nmol (nanomoles) ; kg (kilograms) ; gm (grams) ; mg (milligrams) ; μg (micrograms) ; ng (nanogra s) ;
L (liters) ; ml (milliliters) ; μl (microliters) ; cm (centimeters) ; mm (millimeters) ; jtm (micrometers) ; nm (nanometers) ; V (volts) ; μF (microfarads) and °C (degrees Centigrade) .
In the following Examples, unless otherwise specified, restriction enzymes, T4 DNA ligase, and polynucleotide kinase are obtained from New England
Biolabs. The [α~32P]dCTP, [α~32P]UTP, and [γ-32P]ATP are obtained from Amersham Corporation, and Moloney Murine Leukemia Virus reverse transcriptase buffers, liposome preparations and enzymes are obtained from Bethesda Research Laboratories. These materials are used according to manufacturer's instructions unless otherwise specified. Electroporation is performed using an electroporator from Invitrogen Corporation, and electroporation cuvettes from BioRad Corporation. Oligonucleotides are obtained from Operon Technologies, Inc, or can be synthesized, e.g., on an Applied Biosystems DNA synthesizer according to the manufacturer's instructions. Thermus aquaticus DNA polymerase I is obtained from Perkin-Elmer Cetus. All standard molecular biology techniques are performed according to Sambrook et al . (1989) or Berger and Kimmel (1987), herein incorporated by reference.
Nucleic acid sequences disclosed herein are divided into 10-mer or smaller oligonucleotides as a matter of convenience, and should be interpreted as continuous sequences unless otherwise indicated.
EXAMPLE 1 I. Synthesis of genes encoding tRNA primers The Moloney Murine Leukemia virus (MoMuLV) utilizes the murine tRNA^™ isoacceptor molecules as primers for synthesis of retroviral DNA (Harada, F. , et al . , J. Biol . Chem. 254:10979-10985 (1979); Peters and Dahlberg, J. Virol . 31:398 (1979)). These particular tRNA molecules are found complexed with the MoMuLV reverse transcriptase enzyme in the virion, and this complex, in turn, anneals to the primer binding site on the viral RNA genome (see Figures 2B and 3A) . The MoMuLV reverse transcriptase, capable of utilizing the annealed tRNA molecule as a primer, initiates DNA synthesis, and proceeds to synthesize a DNA molecule that is complementary to the template RNA molecule to which the tRNA primer is annealed.
The nucleotide sequence of murine tRNAproGGG primer is known (Harada et al . , (1979)). Two versions of this tRNA molecule are transcribed in vitro from genetic cassettes which are synthesized using oligonucleotides, then cloned and confirmed by sequence analysis. The deoxyribonucleotide primers used these reactions are depicted below:
Primer No. 1 (SEQ ID No. 1):
5'-ACGGATCCTA ATACGACTCA CTATAGGCTC GTTGGTCTAG GGGTATGATT CTCGCTTGGG GTGCGAG
Primer No. 2 (SEQ ID No. 2) :
5'-TGGAATTCTC TTCATGGGGG CTCGTCCGGG ATTTGAACCC GGGACCTCTC GCACCCCAAG CGAGAA
Primer No. 3 (SEQ ID No. 3):
5'-ACGGATCCTA ATACGACTCA CTATAGAAAA AAATGGTCTA GGGGTATGAT TCTCGCTTGG GGTGCGAG
Primer No. 4 (SEQ ID No. 4): 5'-TGGAATTCTC TTCAAAAAAA AAAAAAAAAA AAAAGAACCC GGGACCTCTC GCACCCCAAG CGAG
Primer No. 5 (SEQ ID No. 5):
5'-AAAAAAAAAA AAAAAAAAAA GAACCCGGG
Primer No. 6 (SEQ ID No. 6) : . 5'-CGAAGCTTTA AAAAAAAAAA AAAAAAAAAG AACCCGGGAC CTCTCGCACC CCAAGCGAG
Oligonucleotide primers No. 1 and No. 2 are designed to produce a recombinant tRNA molecule corresponding to the "wild type" murine tRNAproGGG primer as illustrated in Figure 2A. There are certain differences, in that the in vitro transcribed tRNAs will not contain the modified ribonucleotide bases that exist in their cellular counterparts. However, these modifications have been found to have the same efficacy in terms of both reverse transcriptase-tRNA interaction, and their ability to prime in vitro cDNA synthesis, as the naturally occurring cognate tRNAs (Barat, C. et al . (1989); Weiss, S. et al . (1992); Barat, C. et al . (1991)). The 3'-ends of these primers are complementary such that the 3'-ends of primer No. 1 and primer No. 2 can be annealed in vitro, then treated with the Klenow fragment of DNA polymerase I in the presence of dNTPs to produce a double-stranded DNA molecule comprising a T7 RNA polymerase promoter-tRNAwt encoding cassette (see Figure 2A) . This primer pair is designed such that this bacteriophage T7 promoter sequence is operatively linked to the 5'-end of the tRNA molecule. This promoter-tRNA cassette is flanked with restriction sites to allow the cassette to be cleaved from a cloning vector, and an .Earl restriction site is incorporated into the 3'-end of the cassette so that digestion of the cassette with the Earl endonuclease prior to in vitro transcription reactions results in linearization of the template (hence termination of transcription) at the exact 3'-end of the encoded tRNA sequence. The nucleotide sequences of primer Nos. 1 and 2 are set forth in SEQ I.D. Nos. 1 and 2, respectively. A second primer pair (primer Nos. 3 and 4) is designed to encode a modified form of the tRNA molecule, the modified tRNA molecule being termed tRNApolylJ, as follows: SEQ. ID No. 7
5'-GAAAAAAAUG GUCUAGGGGU AUGAUUCUCG CUUGGGGUGC GAGAGGUCCC GGGUUCUUUU UUUUUUUUUU UUUUUU -45-
As with the tRNAHt, a bacteriophage T7 promoter sequence is operatively linked to the 5'-end of the tRNA sequence and flanking restriction sites and an .Earl site is incorporated. Primer No. 4, encompassing the 3'-end of the tRNApol^j molecule, encodes a poly(U) sequence in place of the 3'-terminal nucleotides of the wild type tRNA molecule (compare the tRNA sequences in Figures 3A and 3B) . Primer Nos. 3 and 4 are set forth in SEQ I.D. Nos. 3 and 4 respectively. Equal molar ratios of primer Nos. 1 & 2, and 3 & 4 are kinased using T4 DNA kinase (polynucleotide kinase) . The kinased oligomer primer pairs Nos. 1 and 2, and primer Nos. 3 and 4 are annealed (80°C for 3 minutes, slowly cooled from 60βC to 37βC over 20 minutes) in Klenow buffer (50mM Tris-chloride (pH 7.6) at 25βC, lOmM MgCl2, lOmM >9-mercaptoethanol) , containing 2mM dNTPs. The annealed primer pairs are then incubated for 30 minutes at 37°C with 10 units of the Klenow fragment of DNA polymerase I in a 30//1 volume to complete double-stranded DNA synthesis. These double-stranded DNA cassettes are then extracted and precipitated using commonly available techniques, resuspended in 1/10 TE buffer (lmM Tris- chloride, O.lmM EDTA (pH 8.0)), and ligated into the dephosphorylated Smal site in the pUClδ cloning vector (Yannish-Perron et al . , Gene 33:103-119 (1985)). The wild type tRNA gene, created by the combination of primer Nos. 1 and 2, is cloned into pUC18 to create pUC18- T7tRNAHt. The modified tRNA gene, created by a combination of primer Nos. 3 and 4, is cloned into pUClδ to create pUC18-T7tRNAPol^J. The sequences and orientations of these two promoter-tRNA cassettes are verified by DNA sequencing, which is performed using Sequenase™ 2.0 (U.S. Biochemicals) in accordance with the supplier's recommendations. -46-
II. Synthesis of tRNA primers Cloned DNA molecules encoding either tRNAHt or tRNApolyU are produced as described above. Transfer RNA molecules are produced from these cloned tRNA genes by incubating the tRNA gene in the presence of bacteriophage T7 RNA polymerase. RNA polymerases generally proceed to synthesize an RNA molecule that is complementary to the template DNA until they encounter a transcription termination signal in the template DNA sequence, or simply run off the DNA template. To ensure that the tRNA molecule produced by the action of the T7 RNA polymerase is properly terminated at the end of the tRNA gene, the tRNA template provided is linearized at the end of the tRNA gene. This can be achieved in a number of ways. One technique involves cutting the cloned insert by restriction enzyme digestion. Digestion with Earl restriction endonuclease removes the promoter-tRNA cassette from pUC18-T7tRNAHt, digesting in vector sequences substantially 5' to the T7 promoter sequence, and digesting precisely at the 3'-end of the tRNA gene template.
Because the Earl restriction site at the 3'-end of the tRNApol^ insert is found to be resistant to digestion, the above approach may not be used with the pUC18- T7tRNApol^j cloning vector. To circumvent this problem an alternative technique, utilizing the polymerase chain reaction (PCR) , is used both to amplify the specific promoter-tRNApol^j fragment, and define the end of the tRNApol^ coding template. For this purpose, two oligonucleotide primers are used: the first primer is a commercially available primer which anneals to sequences in the pUC18 vector located 5' to the inserted T7 promoter region with the 3'-end directed towards the T7 promoter region sequence, and the second is a "Reverse tRNA Primer" (set forth in SEQ I.D. No. 5) which anneals to bases of the desired T7 promoter-tRNAp^ cassette, such that the 5'-most base of the Reverse tRNA Primer is the last 3'-base of the encoded tRNApol^j template, as follows: SEQ. ID No. 8
5'-GAAAAAAATG GTCTAGGGGT ATGATTCTCG CTTGGGGTGC GAGAGGTCCC GGGTTCTTTT TTTTTTTTTT TTTTTT
The PCR is performed according to the following conditions: PCR buffer: 67mM Tris (pH 9.2 at 25βC, 16.6mM (NH 2S04,, 1.5mM MgCl2; 50ng of each primer, approximately lxlO8 molecules of the pUC18-tRNApol^j construct, and 250//M concentrations of each of the deoxynucleotide triphosphates. The reaction mixtures are heated to 100°C for 3 minutes, then cooled to 15°C. The tubes are centrifuged briefly to collect the contents, then 1 unit of Taq polymerase and a drop of mineral oil are added to each 20//1 reaction. The reaction is performed in 40 cycles with the following regimen: 1 minute at 94°C; then 1 minute at 55βC. The amplified products are pooled from 5 reactions, rendered blunt- ended with Klenow fragment and ImM dNTPs, extracted to remove protein and traces of mineral oil, and EtOH precipitated using standard protocols. The pellet is rinsed with 70% ethanol (EtOH) and dried. The pellet is then resuspended in 1/10 TE, and fragments are examined on a 2% agarose/TAE gel against known size standards.
It should be noted that the PCR is known to allow incorporation of single nucleotides (usually deoxyriboadenylic acid residues) to the ends of the amplified product in a template-independent manner
(Clark, J.M., Nucleic Acids Res. 16:9677-9686 (1988)). However, this template-independent incorporation occurs rarely, and when an extra adenine residue is added, it is added to the 3'-end, which is upstream of the T7 promoter, not to the template-encoding sequence. Radiolabeled products of subsequent in vitro transcription reactions utilizing these templates are examined using denaturing polyacrylamide electrophoresis with known size standards, and are of the desired size. Linear DNA fragments containing the T7 promoter- tRNAHt cassette and the T7 promoter-tRNA.,^ cassette are used as the template molecules for the in vitro production of the tRNA molecules. This is achieved by run-off transcription using the bacteriophage T7 RNA polymerase enzyme (see below) .
For in vitro production of tRNAHt, reactions are performed using .Earl-linearized pUC18-tRNAHt template. For in vitro production of tRNApol^j, the PCR-amplified T7-tRNApolyU cassette is used as the template; alternatively, a £>ral sensitive T7-tRNApol^, cassette is linearized and used as template for the in vitro transcription reactions (produced with oligomers Nos. 3 and 6, using the same protocols used for the production of the .Earl compatible T7-tRN pol^j template) . For the in vitro reactions the following conditions may be utilized (Gurevich, V.V. et al . , Anal . Biochem. 195:207-213 (1991) : Briefly, to RNase-free eppendorf tubes the following components are added at 25βC: 80mM Hepes-KOH (pH 7.5), 12mM MgCl2, 20mM DTT, 5mM dNTPs, 2mM spermidine; RNase-free dH20; 50-100 g/ml template DNA and 5//1 of [α_32P]ATP (30/Ci; 3000Ci/mmol; added in order to examine and quantify the products) . The reaction components are mixed, and the reaction is then initiated with the addition of T7 RNA polymerase enzyme reaction mix (to a final concentration of 1200-1800 U/ml) . The tubes are then incubated at 37βC for 4 hours. RNase-free DNase is then added to the reactions, and the digestion of template DNA allowed to proceed for 15-30 minutes; at this point, small aliquots of the reaction mixture can be removed in order to determine the efficiency of incorporation. This determination can be achieved by cold trichloroacetic acid precipitation of an aliquot of the reaction mixture in the presence of an excess of RNase-free carrier DNA. The control for this determination, total counts, is performed on unprecipitated material from the same reaction mixture. To each of the reaction mixtures is then added 150 l of RNase-free dH20 and 20//1 of 3M NaOAc and the mixtures are extracted with an equal volume of phenol/CHCl3 (pH 6.5) (phenol buffered at pH 6.5 is used to minimize the possibility of base-catalyzed hydrolysis of the RNA product) , followed by CHC13. The products are precipitated with 2-propanol, the pellets rinsed, and the precipitate dried. The primer product is resuspended in RNase-free dH20, and an aliquot checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards.
III. Chemical synthesis using solid phase support. The ability to chemically synthesize unmodified oligonucleotides and 2'-0-methyloligoribonucleotides in high yields on solid phase permits the present invention to be readily adapted for use in preparative, analytical and therapeutical applications. Synthetic techniques and reagents useful for modified oligoribonucleotide production (e.g. Sprout, B.S. et al . , Nucleic Acids Res. 17:3373-3386 (1989), incorporated herein by reference), provide a number of embodiments which could not be achieved if the RNA primers are produced in vitro from a DNA template. For example,
2'-0-methyloligoribonucleotides are known to be resistant to RNase activity (Sprout, B.S. et al . , (1989); Inoue, H. et al . , FEBS Lett . 215:327-330 (1987)). Therefore, the use of these modified RNA molecules as primers for an in -50- vivo cDNA synthesis reaction, in place of oligoribonucleotides, results in a resistance to RNase H- catalyzed removal of the initial cDNA synthesis primer (including any existing modifications) . These primers can then be biotinylated, 32P-labeled, or contain sequence elements such as restriction sites which will be copied and incorporated during second-strand cDNA synthesis following base-catalyzed hydrolysis of the original template (the 2'-0"Methyl primer will protect the annealed complementary portion of the original RNA template as wellj . These modifications do not appear to disrupt the ability of the primers to associate with proteins (Sprout, B.S. et al . , (1989)), and the modified oligoribonucleotides anneal with the expected specificity. The elimination of the 2'-OH group renders an RNA primer more resistant to base-catalyzed (nucleophilic) attack on the neighboring 3',5'- phosphodiester bond, and it is resistant to a variety of ubiquitous RNases.
IV. Experimental Design
The modified tRNA primer synthesized as described above is used for in vivo cDNA synthesis reactions in numerous different cell lines. Control reactions are also performed in the absence of primer, with an oligo(dT) primer, or with the in vitro-transcribed wild- type tRNAHt molecule as primer. In these experiments, the primer is introduced into cells via electroporation, along with Moloney Murine Leukemia Virus reverse transcriptase and [α-32P]dCTP. The radiolabeled dCTP is included to facilitate the analysis of the products of these reactions. Care in the preparation of the target cells for the in vivo cDNA synthesis reaction is important, and an assessment of the mode of primer- reverse transcriptase (and, perhaps modified deoxynucleotide triphosphate, or analog) delivery should be carefully considered. Many alternatives exist, as will be obvious to one skilled in the art; some of these techniques are mentioned above, and use of electroporation in the following Examples should not be construed as limiting the scope of the invention.
Following electroporation of the reaction components into target cells and incubation of the cells, the products of the reaction are extracted from the cells by the Hirt extraction technique (Hirt, B. , J. Mol . Biol . 26:365-369 (1967)), and digested to completion with ribonuclease A. Following phenol/chloroform extraction and ethanol precipitation the product is assessed by quantifying incorporation, and by examining of the size of the products separated on agarose gels, with known size standards, using autoradiography. Further, SI nuclease treatment, and RNase H treatment are used to determine the nature of the DNA product.
V. In Vivo cDNA Synthesis in Insect Cells The insect Sf9 cell line is obtained from the
American Type Culture Collection (ATCC) , and maintained as recommended by the supplier. For in vivo cDNA synthesis, the cells are suspended at a concentration of lxlO7 cells/ml in Grace's medium. Prior to electroporation, the reaction components comprising the reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g) and [αr32P]dCTP (50//Ci) are mixed in a total volume of 50//1 in reverse transcription buffer (obtained from BRL) containing dithiothreitol (DTT) (lOmM) , and incubated at room temperature for 10 minutes. The Sf9 cells (5xl06 cells in 0.5ml) are then added to the reaction components and the mixture is transferred into a chilled 0.4cm electroporation cuvette. Electroporation is then performed with the electroporator set at 200V, 250//F and infinite resistance.
Following electroporation, 1ml of warmed Grace's medium is added to the cuvette and the mixture is transferred to a plastic tube (Falcon #2059) . The mixture is then incubated for one hour at 37βC. Although the normal temperature for maintaining the Sf9 insect cell line is approximately 25°C, the obtainment of improved enzyme activity is the primary concern. Following the incubation period at 37°C, the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or equivalent) . The radioactive supernatant is carefully removed and disposed of properly, and 1ml of 0.6% sodium dodecyl sulfate (SDS) , lOmM EDTA (pH 7.5) is added to the cell pellet. Immediately this pellet is gently resuspended with a large bore Pipetman P1000 tip (a portion of the tip removed to increase the bore diameter, thus decreasing the shear forces and fragmentation of genomic DNA) . The viscous lysate is placed on ice for 5 minutes, then 250vl of 5M NaCl is added and the tube inverted several times to mix. The tubes are then placed on ice. Following the incubation period of this Hirt extraction (2-12 hours) , the extract is centrifuged for 20 minutes at top speed in a cold Eppendorf microfuge. The supernatant is carefully withdrawn from the pellet (which is discarded in radioactive waste) , and the supernatant split between two new 1.5ml microfuge tubes. These supernatants are extracted twice with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and 1/6 volume of 2-propanol; the pellets are rinsed with 70% ethanol, and then dried. The pellets are resuspended in 18//1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0) ; the resuspended nucleic acid fraction is treated with ribonuclease A (2/1 of lOmg/ml stock solution), at 37°C for 15 minutes, followed by the addition of 30/1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0). The solution is then extracted once with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and two volumes of ethanol; the pellets are rinsed with 70% ethanol, and dried. Cerenkov counts are obtained on the dried pellets, and the pellets are resuspended and aliquots counted with scintillant. The products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are then stained with ethidium bromide, dried and examined by autoradiography.
VI. In Vivo cDNA Synthesis in Hamster Cells The hamster CHO cell line is obtained from the ATCC, and is maintained as recommended by the ATCC. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO7 or lxlO8 cells/ml. The reaction components are the reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g) , and [α-32P]dCTP (50 Ci) , mixed in a total volume of 50/1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Following preincubation, 0.5ml of the CHO cells are added to the mixture, the cells mixed and immediately transferred to the chilled 0.4cm electroporation cuvette. Electroporation is performed under the following conditions: 330V, 1000//F and infinite resistance. After electroporation, 1ml of warmed, C02- equilibrated Ham's medium (GIBCO) is added to the cuvette, the mixture is transferred to a plastic tube (Falcon #2059) , and the mixture is then incubated for one hour at 37°C.
Following the incubation period, the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent).. The radioactive supernatant is carefully removed and disposed of properly, and 1ml of 0.6% SDS (sodium dodecyl sulfate) , lOmM EDTA (pH 7.5) is added to the cell pellet. Immediately this pellet is gently resuspended with an increased bore Pipetman P1000 tip. The viscous lysate is placed on ice for 5 minutes, then 250 1 of 5M NaCl is added and the tube inverted several times to mix. The tubes are then placed on ice. Following the incubation period of this Hirt extraction (2-12 hours) , the extract is centrifuged for 20 minutes at top speed in a cold eppendorf microfuge. The supernatant is carefully withdrawn from the pellet (which is discarded in radioactive waste) , and the supernatant split between two new 1*5 ml microfuge tubes. These supernatants are extracted twice with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and 1/6 volume of 2-propanol; the pellets rinsed with 70% ethanol, and dried. The pellets are resuspended in 18 1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0); the resuspended nucleic acid fraction is treated with ribonuclease A (2//1 of 10/g/ml stock solution), at 37βC for 15 minutes, followed by the addition of 30/1 of lmM Tris-chloride, O.lmM EDTA (pH 7.0). The solution is then extracted once with phenol/chloroform (1:1), then once with chloroform. The nucleic acid fraction is then precipitated with the addition of 1/10 volume of 3M NaOAc and two volumes of ethanol; the pellets rinsed with 70% ethanol, and dried. Cerenkov counts are then obtained on the dried pellets as described in section V. VII. In Vivo cDNA Synthesis in Human Cells The human HeLa cell line is obtained from the ATCC, and is maintained as recommended by the ATCC. Prior to electroporation, the HeLa cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO7 cells/ml. The reaction components are the reverse transcriptase enzyme (1000 units) , the tRNA primer (5//g) , and [α_32P]dCTP (50//Ci) , mixed in a total volume of 50//1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Then 0.5ml of HeLa cells (5xl06) are added to the reaction components and the mixture is transferred into a chilled 0.4 cm electroporation cuvette (BioRad) . Electroporation is then performed at the following settings: 330V, 1000//F and infinite resistance. After electroporation, 1ml of warmed, C02-equilibrated Eagle's minimal essential media (GIBCO) is added to the cuvette, the mixture is transferred to a plastic tube (Falcon #2059) , and the mixture is then incubated for one hour at 37"C.
Following the incubation period, the cells are subjected to the Hirt extraction as described in section V, and Cerenkov counts are obtained on the dried pellets. The following conditions are used as controls for the in vivo cDNA synthesis reactions in all of the cell lines tested: All reactions contain 50//Ci of [α-3P]dCTP (approximately 3000Ci/mmol) . When included, 1000 units of reverse transcriptase (MoMuLV; BRL) are added to the reaction mixture. In reactions requiring tRNA primer, l-10 g of primer is added per 0.5ml of cells, with the amount determined by the desire to form complexes with all available reverse transcriptase enzyme. The oligo(dT) primer is desirably composed of oligomers of 12-18 bases of deoxyribothymidylic acid; 1 or 5/g of this oligonucleotide primer is added to reactions where indicated.
VIII. Analysis.of Products
As noted in sections V, VI and VII, Cerenkov counts can be obtained on the dried pellets; the pellets can be resuspended (and aliquots counted with scintillant) , and the products electrophoresed on agarose/TAE gels. These gels are run with radiolabeled molecular weight standards, and the gels can be stained with EtBr (see Figures 5, 7, and 9) and dried and examined by autoradiography (see Figures 6, 8, and 10).
The reaction products are treated with SI nuclease to examine the nature of the polydeoxyribonucleotide product(s) . Although SI nuclease will digest a double- stranded nucleic acid molecules to a limited extent, the enzyme works most efficiently on single-stranded nucleic acid substrates; thus polynucleotides which are single- stranded (or contain single-stranded regions) are digested to completion very quickly by SI nuclease treatment (Sambrook 1989) . As is seen in Figure 10, samples treated with SI nuclease, when compared with untreated samples, show no appreciable degradation (Figure 10: compare lanes 2 & 3, and lanes 4 & 5) . Indeed, the prominent cDNA product (approximately 1.9 kb in size) , which appears in the in vivo cDNA synthesis reaction obtained with the tRNAHt primer, shows no appreciable difference before and after SI nuclease treatment (Figure 10: compare lanes 2 & 3) .
To further identify the in vivo cDNA product(s), the SI nuclease treatment is performed on parallel samples before and after ribonuclease H treatment. Ribonuclease H has the activity of digesting the RNA strand of a DNA- RNA heteroduplex. If the double-stranded cDNA product is a heteroduplex, then treatment of the material with ribonuclease H followed by treatment with SI nuclease will result in degradation of the material and loss of an autoradiographic signal in the higher molecular weight range of an agarose/TAE electrophoretic gel. When these experiments are done, the reaction products appear indistinguishable from the products of SI nuclease treatment alone. However, the experiments performed with RNase H and SI nuclease treatment will be repeated in order to rule out heteroduplex in vivo cDNA product.
IX. Cloning in vivo synthesized cDNA products The double-stranded cDNA products from the reactions, above are cloned into vectors using accepted cloning techniques. Briefly, cells are electroporated with the modified tRNA primer, reverse transcriptase enzyme, and [α-32P]dCTP (to follow incorporation) .
Following electroporation and incorporation, the cells are treated as described in Sections V, VI and VII above. The final pellets are rinsed with 70% ethanol, and dried. Cerenkov counts are obtained on the dried pellets, and the pellets are resuspended and aliquots are counted with scintillant. The products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are stained with EtBr (see Figures 5, 7, and 9), dried and examined by autoradiography (see Figures 6, 8, and 10).
It is probable that any second strand cDNA synthesis which occurs with this in vivo cDNA synthesis reaction is primed by the 3'-end of the first strand cDNA product. RNase H activity acts through RNA template degradation to free the 3'-end of the first strand cDNA product and allow flanking sequences to become accessible to annealing by this 3'-end. This is a mechanism common in in vitro cDNA synthesis with this enzyme (Okayama, H. , et al. Recombinant DNA Methodology. Academic Press, Inc., pages 235-260 (1989)). To insure that the ends of the cDNA products are accessible for cloning, the in vivo cDNA reaction products are treated with SI nuclease and rendered blunt-ended with T4 DNA polymerase and high concentrations of dNTPs, prior to the addition of adapters or linker molecules. It is important that SI nuclease be diluted immediately before use, and that the concentration used is determined with small portions of the cDNA product prior to scale-up reactions. Following RNase A treatment, extraction and precipitation, the cDNA is resuspended in SI nuclease buffer: 200mM NaCl, 50mM NaOAc (pH 4.5), lmM ZnS04, 0.5% glycerol and treated with approximately 100 units of diluted SI nuclease per μg of cDNA for 30 minutes at 37°C (Kimmel, A.R. , and S.L. Berger, Guide to Molecular Cloning Techniques , Academic Press, pages 328-329 (1987)). EDTA is added to 20mM to stop the reaction. The products are extracted twice with buffered phenol/chloroform, then once with chloroform. To the tube containing the aqueous phase are added 1/10 volume of sodium acetate and two volumes of ethanol, and the reaction mixture is placed at -70βC for 30 minutes. The tube is centrifuged for 15 minutes in a cold microfuge, and the supernatant is discarded. The pellet is carefully rinsed with 80% ethanol and dried in a speedvac (Savant) .
The product is treated with T4 DNA polymerase in the presence of high levels of dNTPs to insure that the ends are blunted. The cDNA product is suspended in T4 DNA polymerase buffer (50mM Tris-HCl (pH 8.3), 50mM NaCl, lOmM MgCl2, lOmM DTT) . A stock solution containing each of the deoxynucleoside triphosphates is added to achieve a 500//M final concentration. Ten units of T4 DNA polymerase is then added to a final volume of 50//1, and the reaction is incubated at 37βC for 30 minutes. The reaction is stopped with the addition of EDTA to 20mM. Once again, the products are extracted, precipitated, and the pellet rinsed and dried, as described in section V.
Adapters or linkers can now be added to the cDNA product. The use of annealed hemiphosphorylated adapters (Promega; only the 5' blunted end of the adapter is phosphorylated) is considered advantageous in that it allows cloning of the cDNA product after ligation of the adapters and elimination of the excess unligated adapters, without digestion of the cDNA insert to remove linker concatemers. In addition, the step of removing the excess adapters, necessary to eliminate "linker library" construction, can be utilized as well for sizing the cDNA prior to ligation into a vector.
Alternatively, annealed unphosphorylated linkers (Boehringer Mannheim) can be utilized and the excess unligated linkers can be removed simply by heating the ligation product briefly in the range of 60°C to 70°C. This melts the unligated sticky ends, and the ligated, linkered cDNA product can be separated away by column chromatography or gel electrophoresis.
The concentrations of molar equivalent of ends of cDNA and adapters are calculated, and an approximate 20 fold excess of adapters or linkers is used in the ligation reaction (Wu, R. et al . , Meth. Enzymol . 152:343- 349 (1987)). The ligation reaction is performed as follows: to a tube is added 2//1 of 10X ligation buffer (666mM Tris-HCl (pH 7.6), lOOmM MgCl2, lOOmM DTT, 3mM ATP, lOmM spermidine-HCl , lOmM hexaminecobalt chloride, 2mg/ml bovine serum albumin) , hemiphosphorylated adapters, double-stranded, blunted cDNA, and 5 units of T4 DNA ligase in 20//1 total volume. The mixture is incubated overnight at 15βC. The ligation of blunt ends can be enhanced by the addition of PEG 8000; however, polyethylene glycol inhibits phage packaging reactions, and so, should be removed completely prior to these -60- reactions. The ligation reaction is then loaded on a 1ml bed volume Sephacryl S400 spin column (Promega) , which is centrifuged at 800xg to remove excess adapters and adapter dimers. Alternative techniques which allow a more accurate size selection of cDNA product include agarose gel electrophoresis followed by capture and elution from cationic nitrocellulose (e.g. DEAE nitrocellulose) , or recovery directly from harvested sections of low melt agarose. The cDNA is quantified using scintillation, and the molar ends calculated (based on quantity and average size) . The cDNA inserts are now ready for ligation into the vector of choice (constrained somewhat by the size of the inserts and the compatibility of the sticky end) . Once again the ligation is performed as described above. With the use of hemiphosphorylated or unphosphorylated adapters, multiple insert cloning is unlikely, as only the vector has accessible phosphorylated ends.
The ligation reaction is then used for phage packaging, or diluted for chemical transformation or electroporation. Colonies or phage which appear are initially screened with blue/white color selection (alpha complementation) , or with probes to abundant genes. In addition, random colonies or plaques are examined for insert size using PCR with primers which flank the cloning site, or by restriction digest analysis.
Using these general techniques libraries are constructed from cDNA made from CHO cells using the in vivo cDNA synthesis method. One of the libraries is made from the tRNAHt-primed in vivo cDNA product (~1.9kb) from CHO cells (see Figure 8, lanes 4 and 8) which is size- selected on an agarose gel, using the DEAE nitrocellulose (NA45, S&S) capture/elution procedure, prior to cloning. Another library is made from the tRNApol^-primed in vivo cDNA product from CHO cells, which is size selected for cDNA between 4-10 kb using agarose gel electrophoresis.
The cDNA product (-1.9 kb) which appears in CHO cells with the tRNAMt primer may represent sequence- specific priming and synthesis from the conserved, moderately repetitive C-type and intracytoplasmic A-type particle (IAP) sequences found in all CHO cell lines examined (Anderson, K.P. et al . , Virol . 181:305-311 (1991)). These species have extensive homology to the genome of murine leukemia virus.
EXAMPLE 2
I. General Method for assessing suitability of primers for in vivo cDNA synthesis.
Cellular reverse transcriptase cognate tRNA molecules, with modified ribonucleotide bases (see Figure 2B) prime retroviral cDNA synthesis during viral replication. These molecules are not exclusively capable of priming in vivo cDNA synthesis, as evidenced by this invention and inferred from work previously done in vitro (Barat, C. et al . , Nucleic Acids Res. 19:751-757 (1991); Weiss, S. et al . , Gene 111:183-197 (1992); Kohlstaedt, L.A. , and T.A. Steitz, Proc. Natl . Acad. Sci . USA 89:9652-9656 (1992)). In fact, fragments of synthetic tRNA molecules which lack base modifications are capable of annealing to RNA templates in vitro and directing in vitro cDNA synthesis from those templates (Weiss, S. et al . , Gene 111:183-197 (1992)).
In Drosophila , reverse transcription in copia retrovirus-like particles occurs from cleavage and priming from the Drosophila tRNA*et molecule (Kikuchi, Y. et al . , Proc. Natl . Acad. Sci . USA 87:8105-8109 (1990)). Therefore, it is apparent that synthetic polynucleotides which are truncated analogs of a reverse transcriptase -62 - enzyme's cognate tRNA primer molecule(s) may function as primers for this invention as well.
Determination of the suitability of a polynucleotide primer for in vivo cDNA synthesis reaction is straightforward. As defined previously, the primer will meet two (2) important criteria: Any oligonucleotide that is (a) capable of binding in vivo to an RNA template molecule and (b) acting as a primer for at least one reverse transcriptase enzyme such that synthesis of DNA complementary to that RNA template molecule occurs, is a legitimate primer for the in vivo cDNA synthesis of the present invention. Experiments which both quantitatively and qualitatively assess the suitability of a prospective primer may be performed, e.g. , using Chinese hamster ovary (CHO) cells (see Example 1, above) .
The in vivo assays consist of introduction of [α-32P]dCTP, putative primer, and Moloney murine leukemia virus reverse transcriptase into cells via electroporation. Controls for assessing the suitability of primers for in vivo cDNA synthesis can include the following negative controls:
1) Electroporation of [α_32P]dCTP alone.
2) Electroporation of [α_32P]dCTP along with the reverse transcriptase enzyme. An actual primer test reaction can consist of:
3) Electroporation of [α_32P]dCTP along with the "candidate" polynucleotide primer and the reverse transcriptase enzyme.
A positive control reaction can consist of: 4) Electroporation of [α_32P]dCTP along with the modified tRNApol^j primer and the reverse transcriptase enzyme.
The hamster CHO cell line is obtained from the ATCC, and is maintained as described in Example 1. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted, rinsed in PBS and resuspended in PBS at lxlO8 cells/ml. The reaction components include the reverse transcriptase enzyme (1000 units) , the tRNA primer (5 g; or a molar equivalent of the candidate primer), and [α-32P]dCTP (50/Ci) , mixed in a total volume of 50/1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Following preincubation, 0.5ml of the CHO cells are added to the mixture, the cells mixed and immediately transferred to the electroporation cuvette. Electroporation is performed under the following conditions: 330V, 1000//F and infinite resistance. After electroporation, 1ml of warmed, C02-equilibrated Ham's medium (GIBCO) is added to the cuvette, the mixture is transferred to a plastic tube (Falcon #2059) , and the mixture is then incubated for one hour at 37βC.
Following the incubation period, the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) , and subjected to the extraction protocol described in Example 1, Section VI, and Cerenkov counts are obtained on the dried pellets. The pellets are resuspended and aliquots are counted with scintillant. The products are electrophoresed on agarose/TAE gels with radiolabeled molecular weight standards. The gels are then stained with ethidium bromide, dried and examined by autoradiography.
Interpretation of Results
If the results of Cerenkov counts of incorporation of labeled deoxyribonucleotide into the final pellet from the experiment with the candidate polynucleotide primer exceeds the incorporation seen with the negative controls, and the incorporation seen with the positive control is higher than the level seen with the negative control, then the test primer may be priming cDNA synthesis. However, the nature of the cDNA product(s) is very important and should be examined.
The second criteria, therefore, is a qualitative assessment of the product and is determined initially from the pattern and density of the autoradiographic signal obtained from test aliquots run, in parallel with radiolabeled molecular weight standards, on the agarose/TAE gel. If the polynucleotide primer is designed to anneal to an RNA template of heterogeneous size, or to a binding site which is a heterogeneous distance from the 5'-end of the RNA template molecule, then one expects a heterogeneous population of cDNA products. If, however, the primer is designed to anneal to a specific homogeneous RNA template, a specific cDNA product should be produced (compare, for example, Figure 8, lanes 4 and 5; and lanes 8 and 9) . Therefore, both qualitative and quantitative parameters should be examined to determine the efficiency of a prospective primer.
EXAMPLE 3
I. Generation of sequence-specific in vivo cDNA synthesis primers.
The present invention also provides for sequence- specific in vivo cDNA synthesis reactions using a sequence-specific primer which is introduced into cells in combination with a compatible reverse transcriptase enzyme and, if desired, modified deoxynucleotide triphosphate or deoxynucleotide triphosphate analogs. These sequence-specific primers can be synthesized as transcripts prepared from a DNA template or by chemical means (e.g. on solid phase supports) . II. Chemical synthesis using solid phase support. The ability to chemically synthesize unmodified oligonucleotides, and 2'-0-methyloligoribonucleotides in high yields on solid phase permits the present invention to be readily adapted for use in preparative, analytical and therapeutical applications. Synthetic techniques and reagents useful for modified oligoribonucleotide production (e.g. Sprout, B.S. et al . , Nucleic Acids Res. 17:3373-3386 (1989), incorporated herein by reference), provide a number of embodiments which could not be achieved if the RNA primers are produced in vitro from a DNA template. For example,
2'-0-methyloligoribonucleotides are known to be resistant to RNase activity (Sprout, B.S. et al . , (1989); Inoue, H. et al . , FEBS Lett . 215:327-330 (1987)). Therefore, the use of these modified RNA molecules as primers for an in vivo cDNA synthesis reaction, in place of oligoribonucleotides, results in a resistance to RNase H- catalyzed removal of the initial cDNA synthesis primer (including any existing modifications) . These primers can then be fluorpphore- or hapten-conjugated, biotinylated, 32P-labeled, or contain sequence elements such as restriction sites which will be copied and incorporated during second-strand cDNA synthesis following base-catalyzed hydrolysis of the original template (the 2'-0_Methyl primer will protect the annealed complementary portion of the original RNA template as well) . These modifications do not appear to disrupt the ability of the primers to associate with proteins (Sprout, B.S. et al . , (1989)), and the modified oligoribonucleotides anneal with the expected specificity. The elimination of the 2'-OH group renders an RNA primer more resistant to base-catalyzed (nucleophilic) attack on the neighboring 3',5'- phosphodiester bond, and it is resistant to a variety of ubiquitous RNases.
III. Transcription from sequence-specific DNA template. One embodiment capable of producing the desired sequence-specific tRNA primer consists of in vitro transcription from a DNA template, generally as follows:
The initial template for the production of a expression cassette, encoding a sequence-specific tRNA primer, can be the pUC18-T7tRNAHt vector construct. This construct encodes the tRNAwt molecule (see Figures 2B and 3A) . The first of two primers will anneal to sequences which lie in the pUC18 vector 5' to the T7 promoter sequence (see Figure 4) and extend in the 3' direction toward the T7 promoter sequence. A commercially available primer has been used in this manner in the amplification of the pUC18-T7tRNApol^J expression cassette.
The second primer will desirably include bases at the 5'-end of the primer which are complementary to a sequence in the RNA template of the in vivo cDNA synthesis reaction (e.g. an RNA template of known sequence) , but are not complementary to the sequence of the initial DNA template (see Figure 4) . The remaining bases in the primer will be complementary to bases in the DNA template which are a like distance from the precise 3'-end of the encoded tRNA template in the pUC18-T7tRNAHt DNA vector, and extend in the 3' direction toward the T7 promoter sequence (i.e. if 20 bases at the 5'-end of this second primer are complementary to the RNA target for in vivo cDNA synthesis, then the remaining bases will be complementary to bases in the encoded T7tRNAwt DNA starting 20 bases from the 3'-end). This situation is represented in Figure 4. Using these two primers in a PCR, the tRNA primer encoded in the amplified DNA T7 expression cassette is modified during the amplification process, from the initial tRNAHt template, to the desired tRNAspecj ic template (see Figure 4) . Conco itantly, the 5'-base of the second primer defines the 3' boundary of the resultant tRNAspecι-f5c encoding cassette. This cassette produces a tRNAspec1fjc molecule with a 3'-end which is complementary to both the 5'-sequence encoded in the second PCR primer, and the RNA target for the in vivo cDNA synthesis reaction (see Figure 4) .
The PCR is performed in 20/1 volumes according to the following conditions: PCR buffer: 67mM Tris (pH 9.2 at 25°C), 16.6mM (NH4)2S04, 1.5mM MgCl2; 50ng of each primer, approximately lxlO8 molecules of the pUC18-tRNAHt construct, and 250 M concentrations of each of the deoxynucleotide triphosphates. The reaction mixtures are heated to 100βC for 3 minutes, then cooled to 15βC. The tubes are centrifuged briefly to collect the contents, then 1 unit of Taq polymerase and a drop of mineral oil are added to each 20/1 reaction. The reaction is performed in 40 cycles with the following regimen: 1 minute at 94βC; then 1 minute at 55βC. The products are pooled from 5 reactions, blunted with the Klenow fragment, extracted to remove protein and traces of mineral oil, and EtOH precipitated using standard protocols. The fragments are resuspended in lmM Tris- chloride (pH 8.0), O.lmM EDTA and an aliquot is examined on a 2% agarose/TAE gel with known size standards to verify size and to quantify. Typical yields are expected to be approximately 500ng to l//g of product for each 20 1 reaction.
The tRNAspeci ic molecule is then produced in an in vitro transcription reaction in a manner similar to that used for the production of tRNApolyU, above. Briefly, to RNase-free eppendorf tubes the following components are added at 25°C: 80mM Hepes-KOH (pH 7.5), 12mM MgCl2, 20mM DTT, 5mM dNTPs, 2mM spermidine; RNase-free dH20; 50- 100/g/ml template DNA and 5/1 of [α~3P]ATP (30 Ci; 3000Ci/mmol; added in order to examine and quantify the products) . The reaction components are mixed, and the reaction is then initiated with the addition of T7 RNA polymerase enzyme reaction mix (to a final concentration of 1200-1800 U/ml) . The tubes are then incubated at 37°C for 4 hours. RNase-free DNase is then added to the reactions, and the digestion of template DNA allowed to proceed for 15-30 minutes; at this point, small aliquots of the reaction mixture can be removed in order to determine the efficiency of incorporation (This can be achieved by cold trichloroacetic acid precipitation of an aliquot of the reaction mixture in the presence of an excess of RNase-free carrier DNA. The control for this experiment, total counts, is done on unprecipitated material from the same reaction mixture) . To each of the reaction mixtures is then added 150 1 of RNase-free dH20 and 20//1 of 3M NaOAc and the mixtures are extracted with an equal volume of phenol/CHCl3 (pH 6.5), followed by CHC13. The products are precipitated with 2-propanol, the pellets rinsed, and the precipitate dried. The primer product is resuspended in RNase-free dH20, and an aliquot checked for size using autoradiographic exposure of a polyacryla ide/urea gel run with known size standards. The primer is purified and quantified prior to introduction into cells. This approach allows direct amplification of DNA cassettes which are used for in vitro production of sequence-specific tRNA primers for use with the MoMuLV reverse transcriptase enzyme. The technique utilizes a single sequence-specific DNA primer, a 5' universal primer and a tRNAwt DNA template for cassette amplification.
EXAMPLE 4
I. Use of sequence specific primers to determine the ratios of splice variants in cells.
The ability to generate sequence-specific primers for use in the in vivo cDNA reactions enables an investigator to take a real time "snapshot" of the transcription patterns which exist in a target cell or tissue. Care in the preparation of the target cells for this analytical use of the in vivo cDNA synthesis technology is, perhaps, more important than for simple preparative use (e.g. cDNA library construction) , and real assessment of the type of RNA primer, the mode of primer-reverse transcriptase (and, perhaps modified deoxynucleotide triphosphate, or analog) and delivery should be carefully considered. Many alternatives exist for delivering the primer/enzyme complex and a representative selection have been outlined above. One technique for the manufacture of a DNA cassette which allows in vitro production of the sequence-specific primer is outlined in Example 3, above.
Following such synthesis, the primer product is resuspended in RNase-free dH20, and an aliquot checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards. The primer is purified and quantified prior to introduction into cells.
An alternative and more direct approach is to chemically synthesize the RNA primer on solid phase support, generally as described in Example 3, Section II. This approach allows the direct synthesis of the sequence-specific primer, as well as providing an opportunity to incorporate modified stable ribonucleic acid analogs which would be useful for detection, purification and/or modification of the cDNA products (e.g. 32P-labeling, biotinylation, and/or sequence incorporation, respectively) . The cells are prepared in a manner which allows a minimum of perturbation to the desired conditions, and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue. In one such embodiment, the sequence-specific primer, reverse transcriptase enzyme, and [α-32P]dCTP are incubated together briefly in vitro (as described in Example 1, above) , prior to addition to the cells or tissue. Following introduction to the cells or tissue, the cells or tissue is incubated under conditions which permit the synthesis of DNA molecule(s) which are complementary to the specific RNA template. The controls for this reaction include the following: 1) Delivery of [α~32P]dCTP alone. 2) Delivery of [α-32P]dCTP along with the reverse transcriptase. 3) Delivery of [α-32P]dCTP along with in vitro- transcribed "wild type" primer and the appropriate reverse transcriptase enzyme. Following extraction and purification as described, above, the cDNA products can be qualitatively examined by autoradiographic analysis of gels run with appropriate radiolabeled size standards. In addition, the specific products can be quantified by scanning autoradiographs of analytical electrophoretic gels or by liquid scintillation counting of bands excised from preparative electrophoretic gels. The PCR can be used to detect any cDNA which is produced from unprocessed RNA template. EXAMPLE 5
I. Use of multiple sequence specific primers to determine ratios of gene transcripts in cells.
The ability to generate sequence-specific primers for use in the in vivo cDNA reactions also enables one to use a real time "snapshot" of the target cell transcription patterns to detect differential transcription patterns and transcript levels, which are associated with numerous disease states. It is desirable from both a diagnostic and research perspective to identify the levels of various oncogene transcripts, in relation to the levels of an internal control transcript (e.g. a "housekeeping" gene) . PCR has been used for this purpose in the past and is, at best, unreliable and biased due to problems which occur during amplification (Gilliland, G. , et al . (1990)).
Consideration for the use of the invention in this manner, and techniques for the manufacture of DNA cassettes which allow in vitro production of the sequence-specific primers is outlined in Examples 3 and 4, above. A desirable approach in this embodiment is to chemically synthesize the RNA primers on solid phase support, generally as described in Example 3, Section II. This approach allows the direct synthesis of the distinct sequence-specific primers, and provides an opportunity to incorporate distinct modified ribonucleic acid analogs into each primer, which would be useful for differential detection, purification and/or modification of the cDNA products (e.g. 32P-labeling, biotinylation, and/or sequence incorporation, respectively) .
Following such synthesis, distinct probe and control primer products are resuspended in RNase-free dH20, and aliquots are checked for size using autoradiographic exposure of a polyacrylamide/urea gel run with known size standards. The primers are purified and quantified prior to co-introduction into the target cells or tissues.
The cells are prepared in a manner which allows a minimum of perturbation to the desired conditions, and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue.
In one such embodiment, an oncogene sequence- specific primer and an actin sequence-specific primer are utilized as probe primer and control, respectively.
These distinct primers, reverse transcriptase enzyme, and [α_32P]dCTP are incubated together briefly in vitro (as described in Example 1, above) , prior to addition to the cells or tissue. Following introduction, the cells or tissues are incubated under conditions which permit the synthesis of DNA molecule(s) which are complementary to the specific RNA templates. The controls for this reaction include the following: 1) Delivery of [α~32P]dCTP alone. 2) Delivery of [α_3P]dCTP along with the reverse transcriptase. 3) Delivery of [c._32P]dCTP along with the primer for one of the desired RNA templates and the appropriate reverse transcriptase enzyme. 4) Delivery of [α_32P]dCTP along with the primer for each additional desired RNA template and the appropriate reverse transcriptase enzyme. Following extraction and purification as described, above, the cDNA products can be qualitatively examined by autoradiographic analysis of gels run with appropriate radiolabeled size standards. In addition, the identity of specific products can be confirmed using, e.g., biotinylated molecular weight standards, and the gels can be transferred and Southern blots examined using a streptavidin/alkaline phosphatase conjugate system (e.g. the BluGENE System, Bethesda Research Laboratories, Gaithersburg, MD) .
EXAMPLE 6
I. Use of sequence specific primers to clone members of a specific gene family.
The ability to generate sequence-specific primers for use in the in vivo cDNA reactions enables an investigator to selectively produce cDNA from RNA templates which contain a specific sequence. One technique for the manufacture of a DNA cassette which enables production of the sequence-specific primer in vitro is outlined in Example 3, above, and can be used as described. The primer is purified and quantified prior to introduction into cells. A more direct alternative is to use the solid phase chemical synthesis techniques as described in Example 3.
The cells are prepared in a manner which allows a minimum of perturbation to the desired conditions and a delivery system is employed which is assessed to be the best at maximizing the speed of delivery of the reaction components, and minimizing stress to the cells or tissue. Some of these alternatives are outlined in the Detailed Description of the Invention, above.
Initially an analytical protocol is followed. If products of an expected size or size distribution are obtained, then preparative cDNA synthesis reaction(s) can be conducted.
For an analytical assessment of the primers and technique [α-32P]dCTP can be included to rapidly investigate both quantitatively and qualitatively the results of the in vivo cDNA synthesis reaction.
In one experimental scheme, the sequence-specific primer, reverse transcriptase enzyme, and [α-32P]dCTP are incubated together briefly in vitro, as described in Example 1, above, prior to addition to the cells or tissue. Following introduction to the cells or tissue, the cells or tissue is incubated under conditions which permit the synthesis of DNA molecule(s) which are complementary to the specific RNA template. The controls for this reaction might include the following:
1) Electroporation of [α~32P]dCTP alone.
2) Electroporation of [α_32P]dCTP along with the reverse transcriptase. 3) Electroporation of [α~3P]dCTP along with in vitro- transcribed "wild type" primer and the appropriate reverse transcriptase.
Following extraction and purification as described, above, the cDNA products can be qualitatively examined by ethidium stained gels (see Figure 7, lanes 4 and 8), or autoradiographic analysis of gels run with appropriate radiolabeled size standards (see Figure 8, lanes 4 and 8) . In addition, the level of incorporation can be determined by measurement of Cerenkov counts, or, following resuspension, by liquid scintillation counting of aliquots.
Following an analytical examination, similar reactions can be performed in the absence of the radiolabeled deoxynucleotide triphosphates (or with a reduced amount - to use as a tracer) . The number of reactions needed to obtain the desired product(s) is determined by the previous analytical reactions.
The cDNA products from the reactions, above are cloned into vectors using accepted cloning techniques, as described in Example 1, Section IX. EXAMPLE 7
I. Use of sequence-specific primers and four fluorescent dideoxynucleotide analogs for direct sequencing of in vivo cDNA products. A series of fluorescent dideoxynucleotide triphosphate analogs have been developed which have discrete fluorescent emission spectra, and are readily incorporated into DNA by reverse transcriptase enzymes
(Prober, J.M., et al . (1987)). Each analog substitutes for a specific dideoxynucleotide chain-terminating base, and is incorporated into DNA in a template-directed manner. The ability to incorporate modified dideoxynucleotide analogs in vivo, directed from a sequence-specific primer, allows direct sequencing of any RNA template which contains the desired 3'-sequence, and is present in a detectable amount.
There are likely to be some 15,000 different transcripts in a typical eukaryotic cell (Sargent, T.D. (1987) . If a sequence-specific primer is produced with a polyuridylic acid stretch near the 3'-end (designed to position the primer at the polyadenylated 3'-end of the RNA template) , followed directly with a specific 8 base sequence at the extreme 3'-end, then only one cDNA product is likely to be produced (0.258 = 1/65,536) from the mRNA templates present in a eukaryotic cell.
Introduction of this modified tRNA primer (produced in a manner similar to the techniques described in Example 3) , along with the compatible reverse transcriptase enzyme, the bio-11-dUTP deoxynucleotide analog (if the primer is not biotinylated; see Example 3; Example 8, below), and the four (4) fluorescent base- and spectra-specific dideoxynucleotide triphosphate analogs (Prober, J.M. et al . (1987)) results in a nested series of cDNA synthesis products which are extracted and concentrated using, e.g., an avidin-coated polystyrene (Baxter Healthcare, Mundelein, IL) or streptavidin-1inked bead, eluted by heating in gel loading buffer, and electrophoresed on an analytical denaturing polyacrylamide gel. The gel apparatus is attached to a laser-equipped, spectra-discriminating fluorescence excitation and detection device. This allows direct DNA sequencing of expressed genes from an organism without prior cloning, or even preexisting knowledge of sequence. Of course, a similar preliminary experiment (with the inclusion of [α_32P]dCTP, and the absence of the dideoxy chain-terminating analogs) can be performed in order to confirm that a single cDNA species is produced with the primer. This can be confirmed following extraction by autoradiography on an agarose/TAE electrophoretic gel. An alternative technique for obtaining DNA sequence information directly from in vivo cDNA products without cloning is to gel-purify specific cDNA bands on low-melt agarose electrophoretic gels. These products may be directly sequenced using currently accepted sequencing techniques, including solid-phase sequencing if a biotinylated primer is used (Syvanen, A-C. et al . FEBS Lett. 258:71-74 (1989)). The sequence-specific tRNA primer molecule used for cDNA synthesis suggests a sequencing primer which is used for the reaction. Yet another useful approach is to introduce
[α-32P]dNTPs, a biotinylated, sequence-specific tRNA primer, the reverse transcriptase, and one of the four (4) dideoxynucleotide triphosphate chain-terminating molecules into the target cell. In this manner, four separate introductions are performed, each introducing a different chain-terminating dideoxynucleotide base. The cells are then incubated for the time, and in a manner which allows incorporation. The nested cDNA products are extracted and concentrated using, e.g. , an avidin-coated polystyrene (Baxter Healthcare, Mundelein, IL) or -77- streptavidin-1inked bead, and eluted from the bead by heating in sequencing gel loading buffer just prior to loading a sequencing gel. The sequence can then be determined by autoradiography. In addition, these techniques permit a direct approach to determining the 5'-sequence of clones where information concerning the 3'-sequence is available. The choice of a specific 18 base primer sequence will permit specific sequencing in vivo without the time and difficulty of cloning the cDNA product.
EXAMPLE 8
I. Incorporation of biotinylated dUTP during in vivo cDNA synthesis reactions.
The ability to incorporate deoxynucleotide analogs into the in vivo cDNA product allows the preparation and recovery of biotinylated cDNA which can be useful in a number of important areas. Specific biotinylated deoxynucleotide analogs are appropriate for specific experiments, and many candidate substrates exist (Klevan, L. , and Gebeyehu, G. , Meth. Enzymol . 154:561-577 (1987)). The following Example utilizes one approach and should not be construed as defining or limiting alternative methods or techniques for biotinylation of the in vivo cDNA product. Alternatives such as biotinylation of the RNA primer (see Example 3) or incorporation of the dATP analog ^-(-aminoalkylJdATP during the in vivo cDNA synthesis reaction, followed by reaction of the amine- labeled cDNA with a reporter molecule, biotin-N- hydroxysuccinimide ester, after extraction and purification, are useful approaches.
The hamster CHO cell line is obtained from the ATCC, and maintained as recommended. Prior to electroporation, the CHO cells are removed from monolayer culture using a trypsin/EDTA solution. The detached cells are counted. rinsed in phosphate buffered saline and resuspended in phosphate buffered saline at lxlO8 cells/ml. The reaction components are reverse transcriptase enzyme (1000 units) , the tRNA primer (5/g; can be either sequence-specific, or a general primer, e.g. tRNApolyU) , and bio-11-dUTP (0.03mM or 0.3mM final) which are mixed in a total volume of 50/1 in reverse transcription buffer (BRL) containing DTT (lOmM) , and incubated at room temperature for 10 minutes. Following preincubation, 0.5ml of the CHO cells are added to the mixture, the cells mixed and immediately transferred to a chilled 0.4cm electroporation cuvette. Electroporation is performed under the following conditions: 330 volts (V) , 1000 microfarads (//F) and infinite resistance. After electroporation, 1ml of warmed, C02-equilibrated Ham's medium (GIBCO) is added to the cuvette, the mixture is transferred to a plastic tube (Falcon #2059) , and the mixture is then incubated for one hour at 37βC.
Following the incubation period, the cells are pelleted by centrifugation for 5 minutes at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) , and the cDNA product is recovered as described in Example 1. The biotinylated cDNA pellets are resuspended, and the products electrophoresed on agarose/TAE gels. These gels can be run with biotinylated molecular weight standards, and the gels can be transferred and the Southern blot examined using a streptavidin/alkaline phosphatase conjugate system (e.g. the BluGENE System, Bethesda Research Laboratories, Gaithersburg, MD) .
The products can be purified and extracted from preparative agarose gels and used for probes in electron microscopy (using streptavidin-gold) , for in situ hybridization (using streptavidin/alkaline phosphatase detection system (Chan, et al . (1985)), subtractive library construction, as well as other preparative, analytical and therapeutic purposes.
EXAMPLE 9
I. Use of biotinylated in vivo cDNA in subtractive library construction.
The ability to increase the relative frequency of induction-specific, or cell or tissue-specific messages prior to the construction and screening of a cDNA library, is of great strategic benefit, and often the only hope of identification and recovery of genes which are represented by mRNAs of low abundance (Hedrick, S. et al . , Nature 308:149 (1984)). The previously known subtraction techniques utilizes a separation strategy whereby single-stranded cDNA (produced from mRNA selected from the desired cells, tissues or conditions) is annealed to an excess of mRNA extracted from alternate cells, tissues or conditions. The heteroduplex molecules are separated from the single-stranded cDNA (and mRNA) using hydroxylapatite, or other selective matrices. The ability to incorporate deoxynucleotide analogs into the in vivo cDNA product allows the preparation and recovery of biotinylated cDNA.
One of the uses of the biotinylated cDNA product of this in vivo incorporation is in subtractive library construction. This alternative technique of library construction is superior to the prior art, in that both the desired template, and the subtracting template molecules are cDNA, allowing a stability during the hybridization and selection process not previously possible. In addition, the cDNA product is produced from a smaller initial number of cells, with a fidelity and processivity which may exceed the quality of products produced in vitro . This technique relies on the hybridization of biotinylated second-strand cDNA product of one cell population with the cDNA product of first- strand syntheses of the alternately grown or treated cells. Therefore, parameters must be evaluated which yield efficient second-strand products which are biotinylated. Choices in reverse transcriptase enzymes and the like are important, and if the quantity of second-strand product is not sufficient, non-biotinylated in vivo cDNA synthesis may be coupled with second-strand synthesis in vitro (with biotinylated analog incorporation) to augment the amount of this material. The incorporation reaction can be performed as described in Examples 3 or 8, or by alternate means. The biotinylated cDNA is produced in cells or tissue which is grown under conditions other than those of the desired cDNA library. The extracted and isolated biotinylated cDNA product is treated with SI nuclease to cleave any hairpin structure(s) which might link first-strand product from second-strand product; separated from any contaminating sheared genomic DNA by column chromatography or gel electrophoresis, and annealed to approximately 1/30 the quantity of non-biotinylated cDNA product which was produced from the desired cell or tissues, or from cells or tissues grown under alternate, desired conditions (and similarly separated from genomic DNA contaminants) . Routine hybridization conditions are: 120mM NaH2P04 (pH 6.8), 820mM NaCl, lmM EDTA, 0.1% SDS, with the final DNA concentration at approximately 5mg/ml. The reaction mixture is heated to 90°C for 5 minutes, then maintained at 65βC for 12-18 hours. The reaction is diluted in phosphate buffer (120mM NaH2P04, pH 6.8), and the biotinylated cDNA, with annealed common cDNA sequences, is isolated on a streptavidin column. The flow-through cDNA fraction is concentrated by ethanol precipitation and cloned using accepted methods and techniques (Sambrook, (1989)). EXAMPLE 10
I. Incorporation of fluorescent deoxynucleotide analog(s) during in vivo cDNA synthesis directed from an HIV sequence-specific primer to identify HIV-infected CD4 lymphocytes.
There are a number of commercially available fluorescent deoxynucleotide triphosphate analogs which have discrete fluorescent emission spectra and are readily incorporated into DNA by polymerase enzymes. Each analog is incorporated into DNA in a template- directed manner. The ability to incorporate these modified deoxynucleotide analogs in vivo , directed from a sequence-specific primer, allows a rapid and direct method for screening for HIV-infected CD4 lymphocytes using flow cytometry. This screening can be utilized for analytical purposes, or can be used to separate infected from uninfected CD4 lymphocytes, thereby providing a therapeutic approach to HIV-positive individuals.
A human immunodeficiency virus, type I sequence- specific primer for use in the present in vivo cDNA synthesis invention is made with an appropriate HIV- specific sequence. One such PCR primer is prepared, based on the sequence of the SK69 primer (Ou, C-Y et al . , Science 239:295-297 (1988); Zack, J.A. et al . , Cell 61:213-222 (1990)) corresponding to the env region of HIV, as follows (SEQ ID NO. 9):
5'-CTGTTGCAAC TCACAGTCTG GAACCCGGGA CCTCTCGCAC CCC
which can be used in PCR as described in Example 3 to generate a DNA cassette for the production of the HIV- specific tRNA primer.
Alternatively, a PCR primer can be based on the M666 primer of Watson & Wilburn (1992) , corresponding to the U3 region of LAV-1BRU, as follows (SEQ ID NO. 10) : 5'-GGGGAGTGGC GAGCCCTCTT GAACCCGGGA CCTCTCGCAC CCC
The purified, examined and quantitated primer is then introduced into cytokine-activated (Poli, G. and A.S. Fauci, AIDS Res. Hum. Retroviruses 8:191 (1992)) lymphocytes (either fractionated, or unfractionated) utilizing appropriate technique(s) (among them, electroporation and cationic lipid-mediated delivery) , along with MoMuLV reverse transcriptase enzyme, and fluorescent deoxynucleotide triphosphate analog(s) . The cells are incubated under conditions and for a time necessary to produce cDNA to any existing HIV-specific RNA template present in the target cells. A control reaction can be performed on a similar, uninfected cell population It may prove advantageous that many of the fluorophore-conjugated nucleotide analogs undergo a spectral shift upon polymerization. This may allow discrimination between cells in which template-directed cDNA synthesis has occurred, and the background cell population(s) . The cells are washed and then, if desired, incubated on ice for 30 minutes with an antibody directed to the CD4 molecule (e.g. OKT4 from the ATCC) , washed and incubated with FITC-labeled F(ab')2 goat anti-mouse IgG. The cells are then washed again, prepared and screened by Fluorescence Activated Cell Sorting (FACS) using parameters (e.g. gating for cell size, fluorescence and threshold spectra) necessary to excite and screen for the incorporated fluorescent tag(s) . Appropriate parameters can be determined using the negative control cell populations.
If the experiment is done with the inclusion of an anti-CD4 antibody (with appropriate alternate emission wavelength) , these techniques allow both quantitation and separation of HIV-infected lymphocytes. Alternatively, following in vivo cDNA synthesis, the cells can be fixed and permeabilized prior to examination; however, this is primarily an analytical embodiment, rather than a preparative one.
EXAMPLE 11
In Vivo cDNA Synthesis in Monolayer Cell Cultures.
The cells are grown as monolayer culture in 60mm dishes to 60-90% confluence under conditions to maximize transcript populations (e.g. adding interferon to increase the transcription of interferon-induced genes) . The reaction components: the reverse transcriptase enzyme (1000U), the tRNA primer (2-5μg) , and [α-32P]dCTP (50μCi) , mixed in a total volume of 50-100μl in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes. These components are diluted to 0.5ml in serum-free medium containing 50μg of 3:1 (w/w) of DOSPA (2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propanaminiumtrifluoroacetate) :DOPE (dioleoylphosphatidylethanolamine) , a 1:1 (w/w) of DDAB
(dimethyl-dioctadecyl ammonium bromide) :DOPE, or 1:1 (w/w of DOTMA (N-(1,2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride) :DOPE liposomes, and the mixture is incubated at room temperature for 5-30 minutes. An additional 0.5ml of serum-free medium is then added to the mixture. The medium is aspirated off the cell monolayer and the monolayer is rinsed twice with serum-free medium. The reaction mixture is added dropwise to the dish and the dish is immediately rocked to evenly coat the monolayer with the reaction solution. The cells are periodically rocked to insure all areas of the monolayer are evenly coated during a 2 hour incubation period in a 37°C C02 incubator. Three (3) mis of medium containing serum is then added to the dishes, and the dishes returned to the 37°C incubator for 1 hour. Alternatively, cDNA product can be immediately harvested after the 2 hour incubation period following transfer of the radiolabeled reactants to radioactive waste, and rising the monolayer 2X with phosphate buffered saline.
Following the incubation period, the medium is removed from the monolayer, and replaced with 1.5ml of cold medium. The cells are scraped from the dish with a disposable policeman and transferred to microfuge tubes. The remaining cells are rinsed from the dish with 0.5ml of medium which is combined with the cells. The cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) . The radioactive supernatant is carefully removed and discarded properly. A Hirt extraction is carried out on the cell pellets to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions. The cDNA products are then precipitated using standard techniques.
EXAMPLE 12 In Vivo cDNA Synthesis in Suspension Cell Cultures.
The cells are grown in suspension culture and split at regular intervals prior to protocol to insure optimal viability. The reaction components: the reverse transcriptase enzyme (1000U) , the tRNA primer (l-10μg) , and [α-32P]dCTP (50μCi) , mixed in a total volume of 50- lOOμl in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes. These components are diluted to 0.5ml in serum-free medium containing 50μg of 3:1 (w/w) of DOSPA (2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethy1- 1-propanaminiumtrifluoroacetate) :DOPE
(dioleoylphosphatidy-ethanolamine) , a 1:1 (w/w) of DDAB (dimethyl-dioctadecyl ammonium bromide) :DOPE, or 1:1 (w/w/) of DOTMA (N-(1,2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride) :DOPE liposomes, and the mixture is incubated at room temperature for 5-30 minutes. An additional 0.5ml of serum-free medium is then added to the mixture. The cells are pelleted and the medium is aspirated off the cell pellet. The cell pellet is gently resuspended in serum-free medium and pelleted. The medium is aspirated from the cell pellet. The reaction mixture is added to the tube and the tube is immediately flicked to gently resuspend the cell pellet in the reaction solution. The cells are periodically gently flicked to resuspend the cells during a 1 hour incubation period in a 37°C C02 incubator. Three (3) mis of prewarmed and C02-equilibrated medium containing serum is then added to the cells and the cells are transferred to 60mm dishes (Falcon) , and the dishes are returned to the 37"C incubator for 1-4 hours. Alternatively, cDNA product can be immediately harvested after the 1 hour incubation period following transfer of the radiolabeled reactants to radioactive waste, and rinsing the cell pellet 2x with phosphate buffered saline.
Following the incubation period, the cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) . The radioactive supernatant is carefully removed and discarded properly. A Hirt extraction is carried out on the cell pellet to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions. The cDNA products are then precipitated using standard techniques.
EXAMPLE 13 Systemic In Vivo cDNA Synthesis.
Adult mice are obtained from Jackson Labs, maintained under pathogen-free conditions, and provided food and water ad lib. The in vivo cDNA reaction components: reverse transcriptase enzyme (1000U-5000U) , a IgG heavy chain-specific tRNA primer (2-20μg) , and [α-32P]dCTP (50-500) μCi);, are mixed in a total volume of 200μl in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes. These components are diluted to 0.5ml in serum-free medium containing 1 μmol of a 1:1 (w/w) of DOTMA (N- (1,2,3-dioleyloxy)propyl)-N,N,N-trimethy1ammonium chloride) :D0PE liposomes, and the mixture incubated for 5-30 minutes at room temperature. The 0.5ml mixture is slowly injected either into the intraperitoneal cavity or into the tail veins of the mice. The mice are sacrificed 4-24 hours later. The heart, liver, brain, pancreas, and lymph nodes and spleen are removed and weighed. Tissue sections are counted with scintillant and the counts corrected for the weight of the tissue samples. The normalized counts are high in both the spleen and lymph node tissue sections.
In order to visualize the specific in vivo cDNA products, the tissue is gently disrupted, debris is removed and the cells are carried through a Hirt- extraction. The episomal cDNA products are extracted with phenol/chloroform followed by chloroform. The cDNA products are then precipitated using standard techniques. The cDNA products are fractionated (e.g. electrophoresis through a buffered agarose matrix) , and visualized by autoradiography. As an alternative to the use and incorporation of radiolabeled deoxynucleotides, fluorophore- or hapten-conjugated deoxynucleotide analogs can be delivered (or the primer can be labeled) , and the location of the in vivo cDNA products determined by fluorescence of tissue sections (or extracted products) , or by secondary reagents directed at the incorporated analogs.
EXAMPLE 14 Primer-Specific Cell Expression Patterns.
As depicted in Figure 13, a retrovirus reverse transcriptase enzyme complexed with its modified cognate tRNA primer (having a sequence which includes a number of polyuridylic acid residues 5' to a 3'-terminal region containing a specific polynucleotide sequence) will position the enzyme complex on RNA template molecules at the junctional region proximal to the polyadenylic acid stretch present on mature messenger RNAs such that a snapshot of members containing a complementary junctional sequence will be converted to cDNA product.
Stringency in priming and converting mRNA templates to cDNA product will roughly correspond to the statistical formula, 1/4", where n = number of specific bases added to the 3'-end of the polyuridylic acid region of the primer (e.g. if 5 bases are sequence specific following a stretch of 13-15 uridylic acid residues, then 1/45=1/1024 messages would be expected to be primed and converted to cDNA) . If a cell expresses 10,000 different messages, then approximately 10 products would be expected to be primed and converted to a cDNA product with such a primer.
It should be noted that an initial specific polynucleotide template primer molecule may also be used for this purpose, and may prove advantageous in that (1) when an initial specific RNA template molecule is included in the protocol the 5'-region of the initial template molecule can be designated to encode a much more extensive pattern of bases. This will provide an energetically more favorable and potentially more discriminating annealing platform from which priming of cellular messages can occur; and, (2) sequence elements can be included in this initial template primer molecule which will increase the efficiency of initiation of cDNA synthesis.
It is important to note that there are other facts which will affect the resulting primer- and-cell-specific pattern which is generated including RNA template size and frequency, the choice of the reverse transcriptase enzyme and the associated RNase H activity used in the protocol, the choice of nucleic acid analogs incorporated into the cDNA products, the sensitivity of methods used to detect the cDNA products, priming at regions in the mRNA template other than at the 3'-end, the possibility of premature strand transfer reactions, and other factors affecting the efficiency of cDNA synthesis. Following extraction, fractionation, and detection this technology allows a primer-specific cell expression pattern to be developed. When this pattern is generated with a specific primer sequence, the pattern is specific for the type or developmental stage of a cell or tissue. The pattern may be diagnostic for cellular perturbation (e.g. transformation or infection) , or change in response to an applied stimulus. Changes may be characterized solely by changes in the density of various cDNA products which make up the pattern. This pattern technology will be a powerful tool both for diagnostic purposes and for following developmental changes or lineage commitment in cell populations. Fluorophore- or hapten-conjugated deoxynucleoside triphosphate analogs can be used to enhance detection of the in vivo cDNA products. Alternatively, nuclease- resistant (e.g. 2'-0-methyl) and labeled primers may be used for the reactions.
EXAMPLE 15 In Vivo Antisense Therapy.
Directed in vivo cDNA synthesis makes possible the differential conversion of specific cellular transcripts to a form which is unavailable for expression. When specific primer(s) or VCE RNA template primer(s) are introduced into the cells by any of a variety of means (see Examples 1, 11, and 13), or the cells are infected with a defective retrovirus element which includes a template primer molecule whose cDNA product is complementary to the annealing region of the final target polynucleotide molecule (and has complexed within the virus the cognate modified tRNA primer which anneals in the 3'-region of this initial template and a suitable reverse transcriptase enzyme) , the primer-directed conversion of RNA template to cDNA product(s) effectively removes the transcript(s) from the processes required for normal cellular use or expression. In addition, the use of an enzymatic conversion, as opposed to simple hybridization (e.g. antisense nucleotides) or cleavage step (e.g. ribozymes) results in the efficient and stable conversion of template to inert molecular form. This provides an additional advantage, for the products of the antisense reaction can be readily detected, quantified and evaluated. In addition, it is expected that the introduction of a recombinant, defective, defined-tropic virion or particle which contains a VCE template primer molecule with the associated reverse transcriptase enzyme -90- or complex thereof, together with a promoter element (inducible or otherwise) to drive the antisense expression of an included heterologous gene, will provide a vehicle for sustained gene therapy.
EXAMPLE 16
Inclusion of Template Encoded Control Elements in the in vivo cDNA Synthesis Products. During proviral synthesis an initial single-stranded "minus-strand strong-stop" cDNA product is formed when the reverse transcriptase enzyme reaches the 5'-end of the viral genomic RNA template (See Figure 11) . Following removal of the RNA template portion of this cDNA:RNA heteroduplex, the 3'-end of this cDNA product (which includes a sequence made to complement an "R" region near the 5'-end of the viral genomic RNA, "jumps" or anneals to a second "R" region (for repeat) which is located near the 3'-end of the viral genomic RNA segment. cDNA synthesis then proceeds once again, ultimately resulting in replication of the viral genome with terminal sequences which are important for integration into the host genome. An overview of the various steps involved in this process are presented in detail in Varmus, H.E., Science 216:812-821 (1992); Gilboa, E.S., et al . Cell 18:93-100 (1979). The initial steps of this strategy can be utilized by including into an initial RNA template sequences which will be converted to cDNA; then, as in proviral replication, the initial RNA template will be hydrolyzed by the reverse transcriptase-associated RNase H activity, and the remaining single-stranded cDNA will anneal to a polynucleotide template which has a sequence which is complementary to the 3'-end of this initial cDNA molecule. cDNA synthesis will then be resumed and the second template will be covalently joined to the first cDNA transcript within the viable cell. As shown in Figure 12, this strategy allows the intracellular cloning of any available RNA template molecule with the inclusion of desired vector sequences. A region of polyadenylic acid residues at the 5'-end of the first RNA template will result in a polythymidylie acid 3'-region in the initial cDNA product which will anneal to any available polyadenylated messenger RNAs. This allows the in vivo synthesis and construction of a cDNA library. Alternatively, a specific sequence can be included in the 5'-end of this initial RNA template in order to prime cDNA synthesis from specific transcripts. The choice of length and sequence of any polypurine tract is important to preclude the initiation of strand synthesis prior to strand transfer. For the same reason, the choice of the reverse transcriptase enzyme may be important.
The reaction components: reverse transcriptase enzyme (1000U) , a modified tRNA primer (l-10μg) , and [α-32P]dCTP (50μCi) , are mixed in a total volume of 50- lOOμl in reverse transcriptase buffer (BRL) containing DTT, and incubated at room temperature for 10 minutes. The Vector Control Element (VCE) RNA template primer molecule is then added (in an amount dependent upon the size of the template segment) in an additional 30-50//1 of buffer, and the mixture is then incubated at room temperature for an additional 5-10 minutes. This mixture is then electroporated or otherwise introduced into viable eukaryotic cells and the cells are incubated for 1 hour under conditions which allow in vivo cDNA synthesis to occur.
Alternatively, for certain embodiments the modified tRNA primer may be annealed to the VCE RNA template primer molecule prior to the addition of the reverse transcriptase enzyme. However, more often the reverse transcriptase is allowed to form a complex with the modified tRNA primer prior to the addition of the RNA template molecule.
The VCE RNA template primer will have a first sequence to which the modified tRNA primer will anneal and initiate cDNA synthesis, and includes a polynucleotide sequence at the 5'-end which is designed to be complementary and anneals to a 3'-region of a second RNA template molecule. Although the number of 3' nucleotide residues in the modified tRNA primer which anneal to an RNA template primer are constrained due to the size and structure of the primer molecule, the VCE RNA template primer eliminates this constraint. This relaxation in size of the primer complementary region allows very efficient secondary priming of RNA templates with specifically designed single-stranded cDNA primers. In addition, encoded in this initial RNA (GE or VCE) template primer may be one or more of the following: a promoter operatively linked to a gene which confers resistance for biological selection; cre/lox sites, a polycloning site containing restriction enzyme recognition sites; an origin of replication for procaryotic or eukaryotic replication; a promoter which is operatively linked to any cDNA which is inserted 3' to the promoter sequence (or a promoter which is operatively linked to produce an "antisense" message to any cDNA which is inserted) ; transcription enhancer or tissue- specific control elements; and regions which encode products which aid in the detection or recovery of product produced from an inserted sequence (e.g. metal- binding sequences or epitope tags; fusion proteases) . Following the incubation period, the cells are pelleted by centrifugation at setting 5 in an Eppendorf Model 5415C microfuge (or the equivalent) . The radioactive supernatant is carefully removed and discarded properly. A Hirt extraction is carried out on the cell pellet to separate the in vivo cDNA product from the bulk of the genomic cellular DNA. Protein and other contaminants are removed from the cDNA products with phenol/chloroform extractions, followed by chloroform extractions. The cDNA products are then precipitated using standard techniques.
The cDNA products are resuspended and may be subject to directed second-strand cDNA synthesis or other in vitro modification(s) . The final products are ligated under conditions which favor intramolecular ligation (dilute conditions) . The cDNA products are then transformed into bacteria and plated out under standard conditions which favor biological selection for the resistance conferred by the encoded genes, or gene(s) encoded by the VCE RNA template primer molecules.
EXAMPLE 17 In Vivo Transcript Localization. Uridine and deoxyuridine triphosphate conjugates of haptens (e.g. DNP or fluorophores (e.g. fluorescein-12-, tetramethylrhodamine-5-, Texas Red-5-, Cascade Blue-7-,
B0DIPY-FL-X-14-, B0DIPY-TMR-X-14-, and BODIPY-TR-X-14-UTP or dUTP; Molecular Probes, Inc., Eugene, Oregon) serve as substrate analogs of the nucleoside triphosphates, or deoxynucleoside triphosphates, in enzymatic reactions. The deoxyuridine triphosphate analogs can be incorporated into the cDNA during in vivo cDNA synthesis. The choice of deoxynucleotide analog depends on the application; each fluorophore analog has spectral properties (both excitation and emission) which are specific to the fluorophore group and the coupling, and there may be differences in the cytotoxicity or cell inducing properties associated with the various analogs. In addition, each has a membrane partitioning coefficient which may be important in various applications. This latter parameter will affect the substrate availability within the cell or tissue, contribute to the background fluorescence following enzymatic incorporation, and may well limit the choices in the delivery systems. Use of these analogs, or the "caged" analogs described below, in conjunction with a variety of delivery systems allows the in vivo incorporation of cDNA products within cells containing detectable levels of RNA template molecules. This delivery can be to individual cells and tissues, or the components can be introduced systemically.
Sequence-specific primer molecules allow transcript discrimination, and the combined use of radiolabeled primer(s) (or primer(s) labeled with a group which absorbs or emits at a different wavelength) and a second primer(s) introduced with the detectable nucleotide analogs which are incorporated during in vivo cDNA synthesis, will allow dual labeling or differential labeling of cells and tissues. In vivo labeled cDNA products may be detected by any number of means including: FACS analysis of cells; tissue- and animal-sectioning coupled with autoradiography or fluorescence examination; use and detection of secondary reagents directed at incorporated nucleotide analogs; or a combination of these approaches (e.g. autoradiography for one primed cDNA product and fluorescent detection of a second cDNA product, or differential emission spectra to distinguish between cDNA products) . Many of the fluorophore-conjugated analogs undergo a spectral shift upon incorporation into polynucleotides (Molecular Probes, Inc., Eugene, OR) and this may be used to advantage in discriminating between the background signal due to unincorporated substrate from the desired signal from cDNA product - especially if the unincorporated fraction of labeled analogs are not efficiently eliminated from the cell following in vivo cDNA synthesis.
"Caged nucleotides (e.g. the (2-nitrophenyl)-ethyl ester of dATP, Molecular Probes, Inc., Eugene, Oregon) are nucleotide analogs which are released and become available as enzymatic substrates upon photoactivation. Therefore, there are changes in both physiological activity and membrane solubility upon photoactivation. The use of caged nucleotide analogs which are labeled in conjunction with the present cDNA synthesis invention will allow light-directed cDNA synthesis and detection of the resultant cDNA product(s) .
EXAMPLE 18 Retroviral Packaging Cell Lines
Replication-defective retroviruses are used as infective vectors to introduce genes into eukaryotic cells. Typically, sequences containing the gene(s) of interest are packaged into cell lines which express trans-acting functions necessary and lacking in the proviral template. The packaged recombinant template contains obligate cis-acting sequences including 5'- packaging signals (ψ) , direct repeats (DR) , as well as subregions (ψ+) within the gag gene (Armentano, et al . , J. Virol . , 61:1647 (1987); Bender, et al . , J. Virol . , 61:1639 (1987); Adam and Miller, J. Virol . , 62:3802 (1988)). In retroviruses these cis-acting regions appear to be more sensitive to orientation rather than position (Mann and Baltimore, J. Virol . , 54:401-407 (1983); Aronoff and Lineal, J. Virol . , 65:71-80 (1991)), although the efficiency may be affected by signal positioning (Hatzoglou, M. , et al., Human Gene Therapy 1:385-397 (1990). One of the many difficulties associated with infection of cells with replication-defective viruses produced from packaging cell lines is introduction of other heterologous cellular transcripts which were packaged in the packaging cell line.
Heterologous cellular transcripts which may be packaged by a cell line expressing trans-acting functions can be effectively identified and removed by treating the cells with a packaging signal-specific primer sequence (using a modified tRNA primer or an initial VCE RNA template primer) ,. along with a reverse transcriptase enzyme or complex thereof. Alternatively, a primer sequence can be designed to discriminate between the intended target packaging signal, and fortuitous contaminating cellular sequences.
EXAMPLE 19 Gene Therapy
Obstacles to retrovirus-mediated gene therapy include difficulties associated with recombinant virus which are produced in the cell lines which provide trans¬ acting factors which are necessary for packaging the vector. In vivo cDNA synthesis with the Vector Control Element (VCE) RNA template makes possible the in vivo conversion of RNA templates into a proviral form which can be integrated or maintained as an episomal element. Inclusion of promoter elements, centromere elements, telomere elements, origins of replication, as well as other control elements will allow the copy number of an episomal element to be controlled providing long-term meiotic and mitotic stability. EXAMPLE 20 Inclusion of components to Enhance cDNA Synthesis
Inclusion of other components in the reaction mixtures can be useful in many embodiments to enhance the efficiency of product(s) produced with in vivo cDNA synthesis reactions. Retroviral nucleocapsid proteins, as well as other retroviral proteins, have been shown to be significant in the dimerization and packaging of retroviral genomes and may be expected to be useful to optimize efficiency in the present methods as well
(Aiyar, A., et al., J. Virol . 66:2464-2472 (1992); Kahn and Geidroc, J. Biol . Chem. 267:6689-6695 (1992); Prats, A.C. EMBO J. 7:1777-1783 (1988)). The efficacy of any such additional component(s) will be assessed for each embodiment and inclusion will depend to an extent on the enzyme/primer combinations used, and the cell or tissue target of the in vivo cDNA synthesis reaction.
Thus it has been shown that the present invention provides beneficial methods and compositions for cDNA synthesis. It has been shown that in vivo cDNA synthesis reaction works efficiently, as evidenced by: (a) The incorporation of deoxynucleotides into a polynucleotide fraction of a varying size - as is predicted for cDNA synthesis reactions with a heterogeneous mRNA template population (see Figure 6, lanes 5, 9, 11, and 12; Figure 8, lanes 5 and 9) ; (b) The reaction depends on the inclusion of both reverse transcriptase enzyme and primer (Figure 6: compare lanes 3 and 5; lanes 7 and 9; Figure 8: compare lanes 3 and 5; lanes 7 and 9); (c) The reaction products are resistant to ribonuclease A treatment (even when the ribosomal RNA fraction, present in the extracts, is digested to completion (in Figures 5 and 6: compare lanes 5 and 11; lanes 9 and 12) ; and, (d) The heterogeneous nature of the products are dependent on the inclusion of a modified tRNA primer which is designed to anneal and prime off a heterogeneous cellular population of polyadenylated messenger RNA vs. reactions where a single prominent cDNA product is obtained (~1.9 kb) when a specific primer is utilized (Figures 7 and 8: compare lanes 4 and 5; and lanes 8 and 9) .
It has also been shown that the in vivo cDNA synthesis reaction works successfully with a number of unrelated types of cells (In all eukaryotic cells attempted: Sf9 - insect cells; CHO cells - Hamster cells; HeLa cells - Human cells) .
And it has been shown that the products of the in vivo cDNA synthesis reactions appear to be double- stranded polynucleotides - most probably double-stranded DNA, as evidenced by: (a) The treatment with SI nuclease results in no appreciable degradation of the reaction products (see Figure 10: compare lanes 2 and 3; lanes 4 and 5) ; (b) The treatment of the products with ribonuclease H followed by treatment with SI nuclease appears to resuit in no discernible degradation of the reaction products; (c) The in vivo cDNA synthesis reaction does not work when a reverse transcriptase enzyme which lacks ribonuclease H activity is used. The success of the in vivo cDNA synthesis reaction appears to depend, to some extent, on the ability of the reverse transcriptase to digest away the original RNA template; this is a block to second strand synthesis, both in vitro, and in retroviral replication (Tanese, N., et al . , J. Virol . 65:4387-4397 (1991)); and (d) The products are clonable using conventional cloning techniques.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the teaching of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION (i) APPLICANT: Miller, Jeffrey E. (ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR
CDNA SYNTHESIS (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Gray Cary Ware & Freidenrich
(B) STREET: 401 B Street, Suite 1700
(C) CITY: San Diego
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 92101-4297 (V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk
(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release 1.0, Version 1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viϋ) ATTORNEY/AGENT INFORMATION
(A) NAME: Weseman, James C.
(B) REGISTRATION NUMBER: 30,507
(C) REFERENCE/DOCKET NUMBER: P0068US0 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (619) 699 3604
(B) TELEFAX: (619) 236 1048
(C) TELEX: 910-335-1273
(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ACGGATCCTA ATACGACTCA CTATAGGCTC GTTGGTCTAG GGGTATGATT 5 CTCGCTTGGG GTGCGAG 67
(3) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TGGAATTCTC TTCATGGGGG CTCGTCCGGG ATTTGAACCC GGGACCTCTC 5 GCACCCCAAG CGAGAA 66
(4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ACGGATCCTA ATACGACTCA CTATAGAAAA AAATGGTCTA GGGGTATGAT 5 TCTCGCTTGG GGTGCGAG 68
(5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
TGGAATTCTC TTCAAAAAAA AAAAAAAAAA AAAAGAACCC GGGACCTCTC 5 GCACCCCAAG CGAG 64
(6) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AAAAAAAAAA AAAAAAAAAA GAACCCGGG 2
(7) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CGAAGCTTTA AAAAAAAAAA AAAAAAAAAG AACCCGGGAC CTCTCGCACC 5 CCAAGCGAG 59
(8) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: RNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GAAAAAAAUG GUCUAGGGGU AUGAUUCUCG CUUGGGGUGC GAGAGGUCCC 50 GGGUUCUUUU UUUUUUUUUU UUUUUU 76
(9) INFORMATION FOR SEQ ID NO: 8
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 76 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GAAAAAAATG GTCTAGGGGT ATGATTCTCG CTTGGGGTGC GAGAGGTCCC 50 GGGTTCTTTT TTTTTTTTTT TTTTTT 76
(10) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTGTTGCAAC TCACAGTCTG GAACCCGGGA CCTCTCGCAC CCC 43
(11) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGGGAGTGGC GAGCCCTCTT GAACCCGGGA CCTCTCGCAC CCC 43

Claims

Claims: 1. A method for synthesizing a covalently-1inked complementary DNA copy of a plurality of polynucleotide template molecules which method comprises: (a) providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to at least one distinct polynucleotide template molecule;
(b) providing at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template primer molecule;
(c) forming a mixture comprising the reverse transcriptase enzyme or enzyme complex in the presence of the polynucleotide template molecules; and
(d) incubating said mixture under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecules.
2. The method of claim 1 wherein at least one of the polynucleotide template molecules comprises RNA.
3. The method of claim 2 wherein the polynucleotide template primer molecule comprises a modified retroviral RNA template molecule.
4. The method of claim 3 wherein the cDNA product of the modified retroviral RNA template molecule functions as a primer molecule on a messenger RNA template molecule.
5. The method of claim 4 wherein the retroviral
RNA template molecule is modified at its 5'-end with a polyriboadenylic acid sequence.
6. The method of claim 4 wherein the retroviral RNA template molecule comprises a modified Moloney murine leukemia virus RNA template molecule.
7. The method of claim 6 wherein the reverse transcriptase enzyme complex comprises Moloney murine leukemia virus reverse transcriptase and a synthetic reverse transcriptase-cognate primer transfer RNA molecule.
8. The method of claim 7 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule is modified at the 3'-end to contain a sequence other than a sequence which will anneal to the Primer Binding Site.
9. The method of claim 2 wherein the RNA template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule.
10. The method of claim 9 wherein the reverse transcriptase enzyme comprises the cognate reverse transcriptase enzyme.
11. The method of claim 9 wherein the modified hepadnavirus pregenomic RNA template primer molecule is modified at the first region to contain a functional sequence other than the specific binding site sequence epsilon (e) .
12. The method of claim 1 wherein the mixture is formed in a viable target cell.
13. The method of claim 12 wherein the reverse transcriptase enz,yme or complex thereof is introduced into the viable target cell.
14. The method of claim 12 wherein the reverse transcriptase enzyme is synthesized within the viable target cell.
15. The method of claim 12 wherein the reverse transcriptase enzyme or complex thereof and the polynucleotide template primer molecule are introduced separately into the viable target cell.
16. The method of claim 12 wherein the reverse transcriptase enzyme or complex thereof and the polynucleotide template primer molecule are concurrently introduced into the viable target cell.
17. The method of claim 16 wherein the step of introduction is performed by electroporation.
18. The method of claim 12 wherein the viable target cells are eukaryotic cells.
19. A polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a distinct polynucleotide template molecule.
20. The molecule of claim 19 wherein the distinct polynucleotide template molecule comprises messenger RNA.
21. The molecule of claim 19 wherein the polynucleotide template primer molecule comprises a modified retroviral RNA template molecule.
22. The molecule of claim 19 wherein the polynucleotide template primer molecule comprises a modified Moloney murine leukemia virus RNA template molecule.
23. The molecule of claim 19 wherein the polynucleotide template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule.
24. The molecule of claim 19 wherein the second region of said polynucleotide template primer molecule is capable of annealing to a 3' poly (A) tail of a messenger RNA molecule.
25. The molecule of claim 24 wherein said polynucleotide template primer molecule comprises a RNA molecule modified at its 5'-end with a polyriboadenylic acid sequence.
26. The molecule of claim 25 wherein said polynucleotide template primer molecule comprises a modified Moloney murine leukemia virus RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence.
27. The molecule of claim 25 wherein said polynucleotide template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence.
28. A kit for synthesizing a complementary DNA copy of a polynucleotide template molecule, said kit comprising: a) a preparation of a polynucleotide template primer molecule, which molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a distinct polynucleotide template molecule; and
b) a preparation of at least one reverse transcriptase enzyme or complex thereof which initiates transcription from the first region of said template primer molecule and which synthesizes a DNA molecule which comprises regions complementary to said polynucleotide template molecules.
29. The kit of claim 28 wherein said template primer molecule comprises a modified retroviral RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence and wherein the reverse transcriptase enzyme complex is the cognate reverse transcriptase enzyme and the cognate tRNA molecule.
30. The kit of claim 28 wherein said template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence and wherein the reverse transcriptase enzyme is the cognate reverse transcriptase enzyme.
31. The kit of claim 28 wherein the kit further comprises sufficient deoxynucleoside triphosphates to complete the synthesis of at least one complementary DNA copy of the polynucleotide template molecule.
32. The kit of claim 28 wherein the kit further comprises at least one deoxynucleoside triphosphate analog in an amount sufficient to render a resultant complementary DNA molecule capable of being detected.
33. The kit of claim 28 wherein the kit further comprises reagents for introducing the preparations into a viable target cell.
34. A DNA molecule comprising a first DNA sequence encoding a polynucleotide template primer molecule which comprises a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a distinct polynucleotide template molecule.
35. The DNA molecule of claim 35 wherein the DNA molecule further comprises a promoter sequence operatively linked to the 5'-end of the first DNA sequence, said promoter sequence capable of directing transcription of the first DNA sequence.
36. A synthetic polynucleotide template primer molecule the complementary DNA product of which anneals to a distinct polynucleotide template molecule at a position other than a naturally-occurring R region sequence and enables a DNA polymerase enzyme to commence synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecules.
37. A method for synthesizing a complementary DNA copy of a polynucleotide template molecule which method comprises:
(a) providing at least one polynucleotide template molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to said first region, which third region is sufficiently similar in sequence to said second region such that the complementary DNA product of said second region anneals to said third region so as to allow complementary DNA synthesis to recommence;
(b) providing at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template molecule;
(c) forming a mixture comprising the polynucleotide template molecule and the reverse transcriptase enzyme or enzyme complex in the presence of the template molecule; and
(d) incubating the mixture under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecule.
38. The method of claim 37 wherein the polynucleotide template molecule comprises RNA.
39. The method of claim 38 wherein the polynucleotide template molecule comprises a modified retroviral RNA template molecule.
40. The method of claim 39 wherein the retroviral RNA template molecule comprises a modified Moloney murine leukemia virus RNA template molecule.
41. The method of claim 40 wherein the reverse transcriptase enzyme complex comprises Moloney murine leukemia virus reverse transcriptase and a synthetic reverse transcriptase-cognate primer transfer RNA molecule.
42. The method of claim 41 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule is modified at the 3'-end to contain a sequence other than a sequence which will anneal to the Primer Binding Site.
43. The method of claim 38 wherein the RNA template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule.
44. The method of claim 43 wherein the reverse transcriptase enzyme comprises the cognate reverse transcriptase enzyme.
45. The method of claim 43 wherein the modified hepadnavirus pregenomic RNA template primer molecule is modified at the first region to contain a functional sequence other than the specific binding site sequence epsilon (e) .
46. The method of claim 37 wherein the mixture is formed in a viable target cell.
47. The method of claim 46 wherein the reverse transcriptase enzyme or complex thereof is introduced into the viable target cell.
48. The method of claim 46 wherein the reverse transcriptase enzyme or members of the complex thereof are synthesized within the viable target cell.
49. The method of claim 46 wherein the reverse transcriptase enzyme or complex thereof and the polynucleotide template molecule are introduced separately into the viable target cell.
50. The method of claim 46 wherein the reverse transcriptase enzyme or complex thereof and the polynucleotide template molecule are concurrently introduced into the viable target cell.
51. The method of claim 50 wherein the step of introduction is performed by electroporation.
52. The method of claim 46 wherein the viable target cells are eukaryotic cells.
53. A polynucleotide template molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to said first region, which third region is sufficiently similar in sequence to said second region such that the complementary DNA product of said second region anneals to said third region so as to allow complementary DNA synthesis to recommence.
54. The molecule of claim 53 wherein the polynucleotide template molecule comprises a modified retroviral RNA template molecule.
55. The molecule of claim 53 wherein the polynucleotide template molecule comprises a modified Moloney murine leukemia virus RNA template molecule.
56. The molecule of claim 53 wherein the polynucleotide template molecule comprises a modified hepadnavirus pregenomic RNA template molecule.
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57. The molecule of claim 53 wherein said polynucleotide template molecule comprises in the second and third regions sequences other than a naturally- occurring R region sequences and enables a DNA polymerase enzyme to recommence synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecule.
58. A kit for synthesizing a complementary DNA copy of a polynucleotide molecule, said kit comprising: a) a preparation of a DNA molecule which encodes a polynucleotide template molecule, which molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to said first region, which third region is sufficiently similar in sequence to said second region such that the complementary DNA product of said second region anneals to said third region so as to allow complementary DNA synthesis to recommence; and b) a preparation of at least one reverse transcriptase enzyme or complex thereof which initiates transcription from the first region of said template molecule and which synthesizes a DNA molecule comprising regions complementary to said polynucleotide template molecule.
59. The kit of claim 58 wherein said template primer molecule comprises a modified retroviral RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence and wherein the reverse transcriptase enzyme complex is the cognate reverse transcriptase enzyme and the cognate tRNA molecule.
60. The kit of claim 58 wherein said template primer molecule comprises a modified hepadnavirus pregenomic RNA template molecule modified at its 5'-end with a polyriboadenylic acid sequence and wherein the reverse transcriptase enzyme is the cognate reverse transcriptase enzyme.
61. The kit of claim 58 wherein the kit further comprises sufficient deoxynucleoside triphosphates to complete the synthesis of at least one complementary DNA copy of the polynucleotide template molecule.
62. The kit of claim 58 wherein the kit further comprises at least one deoxynucleoside triphosphate analog in an amount sufficient to render a resultant complementary DNA molecule capable of being detected.
63. The kit of claim 58 wherein the kit further comprises reagents for introducing the preparations into a viable target cell.
64. A DNA molecule comprising a first DNA sequence encoding a polynucleotide template molecule which comprises a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a region of the polynucleotide template located 3' to the portion of the template bound by the first region, and a third region located 3' to said first region, which third region is sufficiently similar in sequence to said second region such that the complementary DNA product of said second region anneals to said third region so as to allow complementary DNA synthesis to recommence.
65. The DNA molecule of claim 64 wherein the DNA molecule further comprises a promoter sequence operatively linked to the 5'-end of the first DNA sequence, said promoter sequence capable of directing transcription of the first DNA sequence.
66. A synthetic polynucleotide template molecule the complementary DNA product of which anneals to a second polynucleotide molecule at a position other than a naturally-occurring R sequence and enables a DNA polymerase enzyme to commence synthesis of a DNA molecule which comprises regions complementary to said first and said second polynucleotide template molecules.
67. A recombinant RNA molecule comprising: a modified retroviral template molecule in which sequences comprising the PBS and R sequences are replaced with alternative functional sequence analogs and wherein the molecule functions as a template for in vivo reverse transcriptase polymerase activity.
68. A method for generating a primer-specific cell expression pattern in a viable cell comprising
(a) providing a reverse transcriptase-cognate primer transfer RNA molecule in which the 3' region has been modified to selectively anneal to a subpopulation of cellular polynucleotide template molecules;
(b) providing at least one reverse transcriptase enzyme which initiates DNA synthesis from the first region of the polynucleotide template molecule;
(c) forming a mixture comprising the polynucleotide template molecule and the reverse transcriptase enzyme in the presence of the template molecule in a viable cell;
(d) incubating the mixture under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecule; and (e) analyzing the cDNA products produced in step (d) to produce a primer-specific cell expression pattern.
69. A method for generating a primer-specific cell expression pattern in a viable cell comprising
(a) providing a polynucleotide template primer molecule comprising a first region which binds to at least one reverse transcriptase enzyme or a complex thereof, said first region providing a site where initiation of cDNA synthesis commences, and a second region located 5' to said first region which enables a cDNA product of said molecule to anneal to a subpopulation of cellular polynucleotide template molecules;
(b) providing at least one reverse transcriptase enzyme or complex thereof which initiates DNA synthesis from the first region of the polynucleotide template molecule;
(c) forming a mixture comprising the polynucleotide template molecule and the reverse transcriptase enzyme or complex thereof in the presence of the template molecule in a viable cell; (d) incubating the mixture under conditions which permit the synthesis of a DNA molecule which comprises regions complementary to said polynucleotide template molecule; and
(e) analyzing the cDNA products produced in step (d) to produce a primer-specific cell expression pattern.
70. A recombinant viral particle which is capable of initiating in vivo cDNA synthesis upon introduction into a viable cell comprising a polynucleotide template primer molecule which includes the functional sequence equivalents of the sequence elements required for viral packaging, a region which binds a reverse transcriptase enzyme or complex thereof, and a 5' sequence the cDNA product of which will prime a resumption of cDNA synthesis at a second polynucleotide site.
71. A polynucleotide template molecule which binds at least one reverse transcriptase enzyme or complex thereof and serves as a template molecule for the synthesis of a complementary DNA copy which comprises sequences which encode the genetic elements required for biological selection, replication, and expression of a heterologous gene, and which copy will prime the resumption of cDNA synthesis at least one other site on a polynucleotide molecule.
PCT/US1995/007968 1994-06-22 1995-06-22 METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS WO1995035369A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999013070A1 (en) * 1997-09-11 1999-03-18 Multigene Biotech Gmbh t-RNA PRIMER, PRODUCTION AND USE FOR INHIBITING REVERSE TRANSCRIPTASE
WO2000022114A1 (en) * 1998-10-09 2000-04-20 Ingene, Inc. PRODUCTION OF ssDNA $i(IN VIVO)
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WO2002044421A2 (en) * 2000-11-28 2002-06-06 Promega Corporation Reverse transcription reactions
WO2002044421A3 (en) * 2000-11-28 2003-09-04 Promega Corp Reverse transcription reactions

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CA2193487A1 (en) 1995-12-28
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AU3092195A (en) 1996-01-15

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