WO1997008185A1 - Utilisation d'une amorce de positionnement en 5' pour produire un adn double-brin a partir d'une matrice d'adn - Google Patents

Utilisation d'une amorce de positionnement en 5' pour produire un adn double-brin a partir d'une matrice d'adn Download PDF

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Publication number
WO1997008185A1
WO1997008185A1 PCT/US1996/014046 US9614046W WO9708185A1 WO 1997008185 A1 WO1997008185 A1 WO 1997008185A1 US 9614046 W US9614046 W US 9614046W WO 9708185 A1 WO9708185 A1 WO 9708185A1
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dna
primer
tail
oligonucleotide
strand
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PCT/US1996/014046
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English (en)
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Ike W. Lee
Sanford J. Madigan
George Scheele
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Alphagene, Inc.
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Priority to AU69103/96A priority Critical patent/AU6910396A/en
Publication of WO1997008185A1 publication Critical patent/WO1997008185A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • This invention relates to synthesis of double- stranded DNA.
  • dNTPs deoxyribonucleotide triphosphates
  • poly(rA) herein denotes an RNA homopolymer consisting entirely of adenosine ribonucleotide monophosphate (rAMP) units; “poly(dA)” would refer to a DNA homopolymer having adenosine deoxyribonucleotide monophosphate (dAMP) units.
  • each homopolymer made up of one of the other ribonucleotides or deoxyribonucleotides is herein represented in an analogous manner: e.g., "poly(rC)” is an RNA homopolymer made up of rCMP units; “poly(dC)” is a DNA homopolymer made up of dCMP units; “poly(rG)” is an RNA homopolymer made up of rGMP units; etc.]
  • poly(rA) mRNA species so isolated are then used as templates for the synthesis of cDNA, as follows: first, a synthetic poly(dT)-containing oligonucleotide is hybridized to the 3' poly(rA) tails of the mRNA molecules, where it serves as a primer for synthesis of the first strand of cDNA (sometimes termed the "antisense" strand) by a reverse transcriptase enzyme, using the mRNA as template.
  • this enzyme can synthesize a full-length cDNA strand [i.e., a DNA strand complementary to the entire mRNA template, with the possible exception of some of the poly(rA) sequence at the 3' end of the mRNA], yielding a full-length ss cDNA hybridized to its mRNA template.
  • cDNA strand i.e., a DNA strand complementary to the entire mRNA template, with the possible exception of some of the poly(rA) sequence at the 3' end of the mRNA
  • mRNA*cDNA hybrids may be directly cloned into vectors and used to transform host cells, but the low efficiency of this technique renders it "unsuitable for constructing large numbers of cDNA clones.” (Maniatis et al., p. 221.) More frequently, cloning is accomplished using ds cDNA produced from the ss cDNA by any of several methods, including the following (illustrated in Figs. 1- 4, respectively):
  • ss cDNA The mRNA*cDNA hybrid is first treated with alkali to hydrolyze the mRNA, yielding ss cDNA.
  • ss cDNA will occasionally form, by self-hybridization of a few nucleotides at or near the 3', end of the molecule, transient "hairpin loops" (see Fig.
  • the ss DNA loop joining the two complementary strands of the ds cDNA molecules may be removed with an endonuclease (such as S ⁇ ⁇ nuclease) which specifically digests single-stranded DNA, yielding blunt- ended ds cDNA copies of the original mRNAs, minus varying amounts of each mRNA's 5' terminal sequence.
  • dNTPs i.e., dATP, dTTP, dCTP, and dGTP
  • the polymerase molecule synthesizing the DNA strand primed at the nick nearest to the 5' end of the original mRNA moves along the template, it degrades downstream mRNA fragments and nascent DNA strands, resulting in a ds hybrid having one full-length ss cDNA strand (the first cDNA strand) and a second strand that is partially varying lengths of mRNA (at the 5' end) and partially newly-synthesized DNA.
  • the leftover mRNA portion is removed by residual RNase and the resulting ss DNA tail on the first strand is clipped off, leaving a blunt- ended ds cDNA missing some of the 5' terminal sequence of the original mRNA.
  • the method for ds cDNA synthesis reported by Land et al. produces a ds cDNA copy of the entire mRNA, plus additional sequence that was not originally in that mRNA.
  • the Land et al. method, illustrated in Fig. 3 begins with a ss cDNA from which the mRNA has been removed by alkaline hydrolysis. To the 3' end of this ss cDNA is added a poly(dC) tail, the synthesis of which is catalyzed by the enzyme terminal deoxyribonu ⁇ leotidyl transferase (TdT) .
  • TdT terminal deoxyribonu ⁇ leotidyl transferase
  • This homopolymeric tail at the 3' end of the first cDNA strand then serves as a site for hybridization with a complementary homopolymeric oligodeoxy-nucleotide primer, oligo(dG) .
  • Second-strand DNA synthesis proceeds from the 3' hydroxyl of this primer, yielding a ds cDNA consisting of not only the full-length sequence of the original mRNA, but also a poly(dC-dG) extension at one end. Variations on this method which have been reported include combining a poly(dA) tail with an oligo(dT) primer, or a poly(dT) tail with an oligo(dA) primer.
  • the resulting homopolymeric extension an artifact of the method used to generate the second cDNA strand, remains an integral part of the ds cDNA throughout the cloning procedure.
  • a fourth method reported by Okayama and Berg (Molec. Cellular Biol. 2:161-170, 1982) and illustrated in Fig. 4, also introduces a poly(dG'dC) tail onto one end of the ds cDNA.
  • a ss poly(dT) tail is enzymatically added to the 3 ' end of one strand of a linearized ds DNA vector.
  • Poly(rA) mRNA is hybridized directly to this poly(dT) tail, positioning the 3' hydroxyl of the vector's poly(dT) tail to prime the synthesis of the first cDNA strand along the mRNA template.
  • the 3' end of the newly-formed cDNA strand is then tailed with poly(dC) , and a ds linker containing a ss poly(dG) tail is added to form a bridge between the two ends of the vector (see Fig. 4) .
  • DNA polymerase is employed to generate the second cDNA strand, using the poly(dG) vector tail as primer and the first cDNA strand as template.
  • DNA ligase closes the ds cDNA/vector circle, yielding a recombinant DNA vector containing full-length ds cDNA with a homopolymeric dG-dC extension at the 5' end of the "sense" strand.
  • This method was described by Heidecker and Messing (Nucleic Acids Res. 11:4891-4904, 1983).
  • the invention includes methods for making ds DNAs (including cDNAs and ds DNAs having one or two ss DNA extensions of a defined sequence (e.g., a "sticky end") or a DNA adaptor or ds DNA protrusion) .
  • the positional primer is also useful in a method for comparing the composition of one set of nucleic acids (e.g., mRNAs) with that of another set.
  • the invention features a method for making double stranded (ds) DNA.
  • the method involves the steps of: (a) providing a first DNA strand; (b) adding to the 3' end of the first DNA strand a homopolymeric tail of at least four (preferably at least five) nucleotides, e.g., by ligation or sequential addition of nucleotides, to yield a tailed first DNA strand; (c) contacting the tailed first DNA strand with a single- stranded (ss) primer (the positional primer) which consists of:
  • a second oligonucleotide covalently bonded to the 3 ' terminus of the first oligonucleotide, the second oligonucleotide consi ⁇ ting of at least one deoxyribonucleotide (e.g., one to ten, preferably one to six, and more preferably two to four) complementary to a portion of the first DNA strand immediately adjacent to the 5' end of the tail on the tailed first DNA strand;
  • RNA can be easily removed using either an enzyme such as RNase H or high pH.
  • the ss DNA extension can be removed enzymatically, e.g., by using an appropriate ss-specific DNase.
  • T4 polymerase can be used to remove a 3' ss DNA extension.
  • a 5' ss DNA extension can be removed with an enzyme such as SI nuclease, mung bean nuclease, or Exonuclease VII.
  • a ds RNase such as cobra venom ds RNase
  • Fig. 6 One embodiment of this method is illustrated in Fig. 6, discussed in detail in the Detailed Description section below.
  • non-homopolymeric tail and a complementary, non-homopolymeric oligonucleotide primer, as follows:
  • a first oligonucleotide of at least four nucleotides complementary to all or a 5' portion of the tail (i) a first oligonucleotide of at least four nucleotides complementary to all or a 5' portion of the tail, and (optionally)
  • a second oligonucleotide covalently bonded to the 3' terminus of the first oligonucleotide, the second oligonucleotide consisting of at least one deoxyribonucleotide complementary to a portion of the first DNA strand immediately adjacent to the 5' end of the tail on the tailed first DNA strand;
  • one or both of (1) the first oligonucleotide of the primer, and (2) the tail must include RNA.
  • at least one nucleotide (preferably, the 5' terminal oligonucleotide) of the second oligonucleotide of the primer is not identical to the nucleotide which composes the homopolymeric first oligonucleotide of the primer.
  • the second oligonucleotide of the primer may be omitted, if desired.
  • Such a primer is considered “non- overlapping" because it is not complementary to a portion of the first DNA strand. Nonetheless, such a non- overlapping primer is considered a positional primer because the specific, non-homopolymeric sequence of the primer serves to position the primer on the tail, preferably at a position complementary to the 5' end of the tail.
  • the first DNA strand can include a nucleotide sequence which is complementary to a naturally-occurring mRNA, and the ds DNA can thus be a ds cDNA.
  • the methods described above can thus be used to make a cDNA library; in such a method, the first DNA strand is one of a plurality of first DNA strands, each of which is complementary to a naturally-occurring mRNA.
  • the library of ds cDNAs may be inserted into a cloning vector to create a vectored cDNA library.
  • Another variation on this method permits the preparation of a ds DNA having a ss DNA extension.
  • This method involves the same steps as either of the above methods except that, following removal of the RNA portion of the ds nucleic acid, the resulting ss DNA extension is not removed.
  • either the tail or the portion of the primer complementary to the tail (but not both) is RNA; the other is DNA which is left intact.
  • a non-positional primer to initiate synthesis of the second DNA strand.
  • the primer does not include the second oligonucleotide which is complementary to a portion of the first DNA strand, but rather includes only the first oligonucleotide which is complementary to part or all of the tail.
  • the sequence of the ss DNA extension can be pre-determined by the practitioner by providing a tail having a defined sequence. For example, it may be desirable to have a ss DNA extension which hybridizes to a ss extension produced upon cleavage of a restriction enzyme DNA recognition site by a restriction enzyme. Accordingly, such a sequence may be designed into either the tail or the primer, depending on whether the restriction enzyme of choice produces 5' or 3 ' sticky ends.
  • ds nucleic acid having a ss DNA extension produced by the aforementioned methods is conveniently inserted into a cloning vector having a ss DNA extension which hybridizes to the ss DNA extension of the ds nucleic acid.
  • the invention provides a means for producing a ds DNA having an adaptor.
  • an adaptor consists of a ds portion of at least four (e.g. , five to 50) base pairs and a ss extension of one to 46 (preferably, five to 20) nucleotides.
  • the ss extension of the adaptor allows a ds DNA containing it to be cloned into a vector more efficiently than DNA which is cloned by blunt-end ligation.
  • the ss extension allows a DNA molecule having an adaptor to be cloned in a directional manner.
  • the adaptor is composed of DNA, although RNA adaptors or RNA*DNA hybrid adaptors could be created. Because this method for making DNA having a DNA adaptor does not require RNA, it offers an advantage over RNA-based methods, which require great care to avoid introducing RNases into the reactions.
  • a ds DNA having an adaptor can be made by: (a) providing a first DNA strand (e.g., a cDNA) ;
  • oligonucleotide tail (which can be homopolymeric or non-homopolymeric) of at least four (e.g., five to 50) nucleotides, to yield a tailed first DNA strand;
  • ss primer of at least four nucleotides (e.g., five to 50) complementary to a portion of the tail, provided that the 5' end of the primer does not anneal precisely with (i.e., create a flush end with) the 3' end of the tail;
  • the adaptor may be homopolymeric or non- homopolymeric in sequence.
  • the ss extension of the adaptor may be complementary to a restriction endonuclease "sticky end," in which case, the ss extension is typically four nucleotides in length.
  • the ss extension of the adaptor may be any sequence of one to 46 nucleotides.
  • Fig. 8 Yet another variation on the above methods results in the generation of a ds DNA having a ds protrusion.
  • the method involves the steps of: (a) providing a first DNA strand (e.g., a cDNA) ; (b) adding to the 3' end of the first DNA strand (e.g., by ligation or sequential addition) an oligonucleotide tail of at least four nucleotides (e.g., five to 50), to yield a tailed first DNA strand;
  • a first DNA strand e.g., a cDNA
  • adding to the 3' end of the first DNA strand e.g., by ligation or sequential addition
  • an oligonucleotide tail of at least four nucleotides (e.g., five to 50), to yield a tailed first DNA strand
  • the ds protrusion is composed of DNA, although RNA protrusions or RNA- DNA hybrid protrusions may be made, if desired.
  • the protrusion may be homopolymeric or non-homopolymeric in sequence.
  • the primer and tail may be designed such that the protrusion includes a restriction enzyme DNA recognition site, which may subsequently be cleaved by the restriction enzyme.
  • the second DNA strand can be synthesized in the presence of a methylated nucleotide such as methylated dCTP or methylated dATP. Incorporation of either of these nucleotides can be used to render the synthesized DNA resistant to cleavage by restriction enzymes which recognize dCTP or dATP.
  • the presence of a methylated nucleotide(s) in the synthesized second DNA strand but not in the DNA protrusion allows the DNA protrusion to be cleaved with a restriction enzyme while inhibiting cleavage at recognition sites containing methylated nucleotides.
  • the method of the invention can be utilized for making a ds cDNA library. This method involves:
  • each primer in the set of primers consists of: (i) a first (homopolymeric) oligonucleotide of at least four nucleotides (preferably at least five) complementary to all or a portion of the tail, and
  • a second oligonucleotide covalently bonded to the 3* * terminus of the first oligonucleotide of the primer, where the second oligonucleotide consists of at least one deoxyribonucleotide (e.g., one to ten, and preferably two to five) , and the sequence of the second oligonucleotide is fully degenerate within the set of primers, provided that (A) at least one nucleotide of the second oligonucleotide is not identical to the nucleotide which composes the first oligonucleotide, and (B) one or both of (1) the first oligonucleotide, and (2) the tail, is RNA;
  • non-homopolymeric tails (of at least four nucleotides) and complementary non- homopolymeric first oligonucleotides may be used in lieu of the homopolymeric tails and first oligonucleotides of the primers.
  • non-homopolymeric first oligonucleotide there is no limitation on the extent of the degeneracy of the second oligonucleotide.
  • the homopolymeric or non-homopolymeric tail can include either DNA or RNA.
  • the first oligonucleotide primer must include RNA
  • the first oligonucleotide of the primer includes DNA
  • the tail must include RNA.
  • both the tail and the primer can include RNA unless the method is intended to produce a ss DNA extension on either the sense or antisense strand. Table 1 sets forth some of the various combinations of specific homopolymers and primers which can be used.
  • the corresponding primer is: poly(dC) poly(rG) poly(dG) poly(rC) poly(dA) poly(rU) poly(dT) poly(rA) poly(rC) poly(rG) or poly(dG) poly(rG) poly(rC) or poly(dC) poly(rA) poly(rU) or poly(dT) poly(rU) poly(rA) or poly(dA)
  • the invention also provides a method for making a ds DNA having two ss DNA extensions (i.e., one on each strand) . This, exemplified in Fig.
  • method involves: (a) providing a RNA strand (e.g., a mRNA); (b) adding to the 3' end of the RNA strand a RNA tail of at least four (preferably at least five) nucleotides, e.g., by ligation or sequential addition of nucleotides, and adding to the 5' end of the RNA strand a RNA leader of at least four (preferably at least five) nucleotides, e.g., by ligation of an oligonucleotide, to yield a RNA strand having a tail and a leader;
  • a RNA strand e.g., a mRNA
  • a primer which is complementary to a portion of the RNA is used to initiate synthesis of the first DNA strand.
  • the first primer used can be thought of as consisting of two oligonucleotides; here, the first oligonucleotide of the primer is RNA and is complementary to all or a portion of the RNA tail.
  • the second oligonucleotide is covalently bonded to the 3* * end of the first oligonucleotide.
  • This second oligonucleotide is DNA and is complementary to a portion of the first RNA strand immediately adjacent to the 5' end of the tail on the tailed first RNA strand.
  • the RNA portions of the ds nucleic acid may be removed with high pH, or with an RNase specific for the RNA portion of an RNA-DNA hybrid (e.g., RNase H) .
  • the RNA tail and leader may be homopolymeric or non- homopolymeric.
  • the RNA tail and the RNA leader may be identical in sequence to each other or they may have different sequences. Because the sequence of the tail and leader can be pre-determined, the ss DNA extensions can be designed to be complementary to ss DNA extensions produced by restriction enzymes.
  • the RNA tail and RNA leader may be added to the mRNA simultaneously or, if desired, they may be added sequentially.
  • the invention also features a method for comparing the composition of a first population of RNA molecules with the composition of a second population of RNA molecules (for example, comparing the mRNA species present in one biological sample with those present in a second sample) .
  • This method can be employed to identify a gene which is expressed differentially (i.e., to a greater extent) in one cell type versus in another. It is thus a modification of a "differential display" technique.
  • This method exemplified in Figs. 12a and 12b, includes:
  • RNA molecules e.g., mRNA
  • RNA molecules e.g., mRNA
  • second population of DNA strands complementary to the second population of RNA molecules e.g., mRNA
  • each primer in the first set consists of (i) a first (homopolymeric) oligonucleotide of at least four (preferably four to 50) nucleotides complementary to all or a portion of the tail, and
  • nucleotide sequence of the second oligonucleotide is fully degenerate within the first set of primers, provided that (A) at least one nucleotide of the second oligonucleotide is not identical to the nucleotide which composes the homopolymeric first oligonucleotide, and (B) one or both of (1) the first oligonucleotide, and (2) the tail, is RNA;
  • ss DNA or less preferably, RNA
  • primers preferably seven to 15 nucleotides; more preferably eight to 12; most preferably nine to 11
  • the second set consisting of oligonucleotides with one or a plurality of sequences
  • a second oligonucleotide covalently bonded to the 3 ' terminus of the first oligonucleotide, the second oligonucleotide consisting of one or more (preferably three or two) deoxyribonucleotides, wherein the nucleotide sequence of the second oligonucleotide is fully degenerate within the first set of primers, provided that one or both of (A) the first oligonucleotide, and (B) the tail, is RNA;
  • ss primers preferably seven to 15 nucleotides; more preferably eight to 12; most preferably nine to 11
  • the second set consisting of oligonucleotides with one or a plurality of sequences
  • the above-described methods for comparing two populations of RNA molecules can also include, prior to the comparing step, separating the species of the first population of ds DNAs (e.g., by size, using high resolution polyacrylamide gel electrophoresis) to produce a first characteristic pattern; and separating the species of the second population of ds DNAs to produce a second characteristic pattern.
  • an "oligonucleotide” may consist of only a single nucleotide, or it can include more than one nucleotide. Unless further defined, it can be either RNA or DNA.
  • homopolymeric is meant composed of a single type of nucleotide (e.g., dC, rC, dG, rG, dA, rA, dT, or rU).
  • a nucleotide sequence which is "complementary" to a reference sequence is capable of base-pairing with every nucleotide of the reference sequence.
  • a homopolymeric oligonucleotide consisting of poly(dC) is complementary to a homopolymeric oligonucleotide consisting of poly(rG) .
  • the invention offers several advantages over most previously-described methods of synthesizing ds DNA.
  • a primer which hybridizes to the 3* * tail and which is positioned such that deoxyribonucleotide(s) of the primer hybridize precisely with the 3' end of the non- tail portion of the first DNA strand, and by requiring that at least one of (i) the tail and (ii) the primer be RNA, the invention permits the formation of a full-length ds DNA with no extraneous tailing to interfere with expression.
  • the invention does not result in the loss of any sequence corresponding to the 3 ' end of the first DNA strand (or the 5*" end of the original RNA template, if the first DNA strand was generated by reverse transcription of an RNA such as mRNA) .
  • RNA such as mRNA
  • the full-length ds cDNA that is generated by the method of the invention preserves all or nearly all of the 5' untranslated region present on the original mRNA, which is potentially necessary for full expression efficiency of the cloned cDNA.
  • the resulting full-length ds cDNA can be cloned and expressed with high efficiency.
  • Variations on the method of the invention produce ds DNAs having one or two defined single-strand extensions. These DNAs can readily be cloned into appropriate cloning vectors having ss DNA extensions complementary to the ss extension(s) on the ds DNA. Because the sequence of the ss DNA extension(s) can be chosen, the ds DNA can be engineered to have, for example, a ss DNA extension(s) which is capable of hybridizing to a ss DNA extension produced by a restriction enzyme. Such a DNA molecule can be inserted into a cloning vector more efficiently than blunt-ended DNAs or DNAs to which linkers, tails, or adaptors are ligated.
  • the ss DNA extension(s) allows the DNAs to be cloned in a directional manner. It is expected that where a ss DNA extension is left on the ds DNA, it will be relatively short (e.g., less than ten nucleotides and preferably about four or five nucleotides) in order to maximize the efficiency of cloning and expression and minimize experimental artifacts, such as those observed by Xu et al. (Xu et al., DNA 6:505-513, 1987).
  • FIG. 1 is an illustration of a traditional method of preparing ds cDNA.
  • Fig. 2 is an illustration of a second traditional method of preparing ds cDNA.
  • FIG. 3 is an illustration of a third traditional method of preparing ds cDNA.
  • Fig. 4 is an illustration of a fourth traditional method of preparing ds cDNA (adapted from Fig. 2 of Okayama and Berg, p. 163) .
  • Fig. 5 is an illustration of a fifth method of preparing ds cDNA, as disclosed in Fig. 5 of Scheele et al., U.S. Patent No. 5,162,209.
  • Fig. 6 is a illustration of one embodiment of the invention. The illustrated method is used to prepare full-length ds DNA using a homopolymeric tail.
  • Fig. 7 is a illustration of one embodiment of the invention. The illustrated method is used to prepare full-length ds DNA using a non-homopolymeric tail.
  • Fig. 8 is an illustration of an embodiment of the invention. The illustrated method is used to prepare full-length ds DNA having one ss DNA extension.
  • Fig. 9 is an illustration of an embodiment of the invention. The illustrated method is used to prepare full-length DNA having an adaptor.
  • Fig. 10 is an illustration of an embodiment of the invention. The illustrated method is used to prepare full-length ds DNA having a ds protrusion.
  • Fig. 11 is an illustration of a method of the invention which is used to prepare full-length ds DNA having two ss DNA extensions.
  • Figs. 12a and 12b are an illustration of a method of the invention which is used to compare the compositions of two populations of RNA molecules.
  • the method illustrated in Fig. 6 involves the following steps:
  • a DNA strand which may be, for example, denatured genomic DNA, a DNA virus, or DNA synthesized from an RNA template (such as mRNA or an RNA of a virus)].
  • the 3' terminal nucleotide of the DNA happens to be dA.
  • a homopolymeric oligonucleotide tail is then added to the 3' end of the DNA strand with TdT and dCTP (a non-homopolymeric oligonucleotide may be added in an alternative method) .
  • the tail can be added to the DNA strand while the DNA strand is hybridized to a second nucleic acid (such as a second DNA strand or an RNA (e.g., a mRNA)). If the DNA strand is hybridized to a second nucleic acid, conventional methods can be used to remove the second nucleic acid after the tail is added onto the first DNA strand. Alternatively, the first DNA strand can be in single-stranded form when the tail is added (as is illustrated in Fig. 6) .
  • a second nucleic acid such as a second DNA strand or an RNA (e.g., a mRNA)
  • the tail is generally attached to the 3' end of the DNA strand via sequential addition of nucleotides by an enzyme such as TdT or TdT used sequentially with poly(A) polymerase.
  • an enzyme such as TdT or TdT used sequentially with poly(A) polymerase.
  • one to three ribonucleotides can be added to the 3' end of the first DNA strand in a reaction which includes approximately 5 ⁇ g DNA, a nucleotide at a concentration of l mM, and 10 units of TdT in a conventional buffer. Such a reaction may proceed at 37° C for approximately three hours.
  • the tail can be extended further in a reaction employing poly(A) polymerase.
  • RNA having an RNA tail e.g., DNA produced in the TdT reaction described above
  • DNA having an RNA tail can be extended in a reaction employing 10 units of poly(A) polymerase, and a nucleotide at a concentration of 1 mM.
  • the reaction is optimized to produce a DNA strand having a tail of 20 to 40 nucleotides.
  • ligation of a suitable pre-formed homopolymeric or non-homopolymeric oligonucleotide to the 3 ' end of the DNA strand would also suffice.
  • a suitable pre-formed homopolymeric or non-homopolymeric oligonucleotide may contain 10 units of T4 RNA ligase, 20 nM of the oligonucleotide (which should have a 5' phosphate group, as is shown in Fig. 7), and 10% dimethyl sulfoxide in a buffer (e.g., 50 mM HEPES, pH 7.5; 20 mM MgCl 2 ; 3 mM DTT; 0.1 mM ATP; and 10 ⁇ g/ml bovine serum albumin) .
  • a buffer e.g., 50 mM HEPES, pH 7.5; 20 mM MgCl 2 ; 3 mM DTT; 0.1 mM ATP; and 10 ⁇ g/ml
  • RNA ligase may be used to add a ribonucleotide having both a 5' phosphate and a 3' phosphate to the 3' end of the first DNA strand.
  • the 3' phosphate should subsequently be removed (e.g., with bacterial alkaline phosphatase) .
  • RNA is better than DNA as an acceptor of a RNA tail (i.e., as a donor) added by RNA ligase; an oligonucleotide consisting of rA is the most efficient acceptor.
  • RNA tail is added by RNA ligase
  • TdT to add several rNTPs to the 3' terminus of the first DNA strand.
  • RNA ligase can also be used to ligate a DNA tail to the 3* * end of the first DNA strand.
  • donors i.e., tails
  • DNA and RNA are equally reactive.
  • the tail may be DNA [poly(dC) , poly(dG) , poly(dA) , poly(dT) , or an oligonucleotide having a mixture of deoxyribonucleotides] or RNA [poly(rC) , poly(rG), poly(rA) , poly(r ⁇ ) , or an oligonucleotide having a mixture of ribonucleotides] .
  • a poly(dC) tail has been shown to offer some advantages over a poly(dG) tail, which in ss form can condense into a non-productive secondary structure that produces experimental artifacts (such as difficulty in hybridizing to a complementary primer) ; additionally, the rate of tail synthesis by TdT (and thus tail length) is more readily controlled by time and temperature manipulations when dCTP is the nucleotide used than when dGTP is the nucleotide.
  • a poly(dC) tail offers an advantage over poly(dA), poly(dT), poly(rA) and poly(rU) tails because the three hydrogen bonds formed in each G-C pair provide more hybridization stability than do the two hydrogen bonds formed between A-T or A-U pairs.
  • a poly(dC) tail means that the primer could be poly(rG) , which is more resistant to contaminating RNase A than would be a primer of poly(rC) or poly(rU) . It is most preferred that the tail be composed of poly(dA) , poly(dT), or poly(rU) because the tail and primer are thus less likely to form an obstructive secondary structure than are tails and primers composed of poly(dC) and poly(dG).
  • the precise length of the tail is not critical, provided that it is long enough to hybridize efficiently and stably with the primer (i.e., at least four nucleotides long) . A range of approximately 4 to 50 nucleotides is recommended for the length of the tail.
  • a single-stranded primer complementary to all or a portion the tail and a portion of the first DNA strand is then allowed to hybridize with the tail and a portion of the first DNA strand.
  • the primer consists of two portions, for convenience denoted the “first oligonucleotide” and the “second oligonucleotide.”
  • the first oligonucleotide is complementary to all or a portion of the tail. Where a homopolymeric tail is used, the first oligonucleotide of the primer is therefore also homopolymeric.
  • the first oligonucleotide of the primer is obviously non- homopolymeric, and the second oligonucleotide may be omitted, if desired.
  • the primer is considered an "overlapping" primer because it anneals to a portion of the non-tail DNA as well as to part or all of the tail.
  • This first oligonucleotide of the primer consists of at least four nucleotides. In practice it is unlikely to be longer than approximately 50 nucleotides, and in fact there is probably no reason to make it longer than ten, or even five nucleotides.
  • the first oligonucleotide of the primer can consist of either ribonucleotides or (where the tail consists of RNA) deoxyribonucleotides. If desired, the first oligonucleotide of the primer may consist of a mixture of ribonucleotides and deoxyribonucleotides. In such a case, the tail must also consist of a combination of ribonucleotides and deoxyribonucleotides such that the deoxyribonucleotides of the tail and of the first oligonucleotide of the primer are hybridized only to ribonucleotides and not to other deoxyribonucleotides. The tail and primer may subsequently be removed with a combination of RNases and DNases used simultaneously or sequentially.
  • the second oligonucleotide of the primer is covalently linked to the 3 ' end of the first oligonucleotide. It consists of one or more deoxyribonucleotides which are complementary to a portion of the first DNA strand immediately adjacent to the 5' end of the tail on the tailed first DNA strand. This permits the primer to hybridize to the tailed first DNA strand at a point that precisely straddles the junction of the 5 ' end of the tail and the 3' end of the non-tail portion of the first DNA strand (see Fig. 6) , rather than at random places anywhere along the length of the tail.
  • the primer is considered a "hybrid" primer because it is a hybrid of RNA and DNA, as the second oligonucleotide is always DNA.
  • a set of hybrid primers 30 nucleotides in length with each primer having a first oligonucleotide composed of poly(rA) and a second oligonucleotide composed of two nucleotides which are degenerate in sequence (excluding dA at the 5* * terminal position) , is preferred.
  • a hybrid primer is employed in the example illustrated in step (b) of Fig. 6.
  • the first oligonucleotide of the primer in this example is homopolymeric and consists of RNA (5'rGrGrGrG3') which is complementary to the tail.
  • the second oligonucleotide in this example consists of a single deoxyribonucleotide (dT) , and this oligonucleotide is complementary to the nucleotide immediately adjacent to the tail on the first DNA strand (dA) .
  • the 3* * terminal deoxyribonucleotide of the first DNA strand (the nucleotide to which the tail is attached) is of the same type as the nucleotide used to form the homopolymeric tail (e.g., where the tail is poly(rA) or poly(dA) and the 3' terminal deoxyribonucleotide of the first DNA strand is dA, or where the tail is poly(rU) or poly(dT) and the 3' terminal deoxyribonucleotide is dT) .
  • the first oligonucleotide of the primer is a homopolymeric oligonucleotide
  • at least one nucleotide (preferably the 5' terminal nucleotide) of the second oligonucleotide of the primer should not be of the same type as the nucleotide which composes the homopolymeric oligonucleotide of the primer.
  • the sequence and type of the first oligonucleotide included in the primer are, of course, dependent upon the sequence and type of nucleotides of the tail. Some of the possible tail/primer combinations are shown in Table l above.
  • the primer may be of synthetic or natural origin.
  • the length of the primer is not critical, provided that it is long enough to hybridize efficiently and stably with all or a portion of the tail and with a portion of the first DNA strand (i.e., the entire primer should be at least five nucleotides long) .
  • a range of 5 to 60 nucleotides is recommended for the primer, preferably with approximately four to 20 nucleotides in the first oligonucleotide and approximately one to ten in the second.
  • either (i) the tail or (ii) the first oligonucleotide of the primer (or both) must be RNA.
  • the first oligonucleotide of the primer may be either RNA or DNA; if the tail is DNA, the first oligonucleotide of the primer must be RNA.
  • the primer may be shorter than, longer than, or the same length as the tail.
  • RNA is notoriously sensitive to degradation by ubiquitous RNases. Therefore, great care must be taken in handling primers and tails that include RNA, in order to prevent contamination with RNase and loss of the primer or tail. Even where the usual precautions are taken, a stored preparation of RNA primer or tail can be expected to be gradually degraded, and thus to lose (over a period of months) its effectiveness as a primer or tail for DNA synthesis.
  • the second DNA strand is synthesized by adding DNA polymerase and all four standard dNTPs (or conventional, modified dNTPs) to the primed template (the arrow at the end of the synthesized DNA strand indicates the direction of synthesis) .
  • the first (or first several) deoxyribonucleotides of the second DNA strand are provided by the deoxyribonucleotides of the second oligonucleotide of the primer (see Fig. 6; step (c)).
  • RNA either the first oligonucleotide of the primer or the tail or both
  • an appropriate enzyme e.g., RNase H where only one of the tail or the first oligonucleotide of the primer is RNA (as in Fig. 6, step (d))
  • a ds RNase such as cobra venom ds RNase
  • the first oligonucleotide of the primer or the tail is DNA, it will thereupon be rendered single- stranded, and can conveniently be removed using an appropriate ss DNA nuclease: e.g., T4 polymerase will remove a 3' ss DNA extension, as where the tail is DNA (as in Fig. 6, step (e)) , while other nucleases (such as SI nuclease, mung bean nuclease, and Exonuclease VII) are useful for removing a 5* * ss DNA extension, as where the entire primer is DNA.
  • T4 polymerase will remove a 3' ss DNA extension, as where the tail is DNA (as in Fig. 6, step (e))
  • other nucleases such as SI nuclease, mung bean nuclease, and Exonuclease VII
  • ss DNA extension left after removal of the RNA primer or tail results in a blunt-ended, full-length ds DNA suitable for further use, including cloning.
  • the invention is useful for making full-length ds DNA from a variety of sources of ss DNA, such as ss DNA viruses, any denatured DNA, or reverse transcripts of RNA viruses and other RNAs.
  • the methods of the invention may be used to convert any ss DNA strand into a full-length ds DNA, even if the sequence at the 3' end of the ss DNA strand is unknown.
  • the invention has wide applicability in the field of cDNA cloning, as it permits cloning of a ds cDNA representing the entire mRNA sequence, including the entire 5*" untranslated region.
  • the invention can avoid introducing into the clone long stretches of homopolymeric DNA upstream of the gene, which may interfere with cloning and expression of the cDNA.
  • This example provides a detailed protocol for using the method of the invention to construct a cDNA library from RNA having a poly(A) tail (e.g., mRNA).
  • RNA having a poly(A) tail e.g., mRNA
  • modifications of this protocol e.g., "scaling up" may be made in order to optimize the method for a particular RNA sample.
  • 5 ⁇ g of poly(A) RNA are combined with 2.5 ⁇ g of poly(dT) a
  • HindIII-XhoI-(dT) 23 primer Such a primer has Hindlll and Xhol sites at its 5* * end.
  • the final volume should be 30 ⁇ l in water or a suitable buffer (e.g., a Hepes buffer).
  • the sample is heated to 70°C for approximately 8 minutes and then chilled on ice for 2 or more minutes. These steps of the method produce an annealed poly(A) RNA/primer.
  • the annealed poly(A) RNA/primer is then incubated at 45° C for 1 hour in the presence of 10 ⁇ l of a conventional 5X reverse transcriptase reaction Buffer, 5 ⁇ l of 100 mM DTT, 5 ⁇ l of 5 mM of a mixture of dNTPs, and 200 U of reverse transcriptase (e.g., SUPERSCRIPTTM (Life Technologies, Inc.)).
  • a conventional 5X reverse transcriptase reaction Buffer 5 ⁇ l of 100 mM DTT, 5 ⁇ l of 5 mM of a mixture of dNTPs, and 200 U of reverse transcriptase (e.g., SUPERSCRIPTTM (Life Technologies, Inc.)
  • the reaction may be labeled for subsequent analysis (e.g., by gel electrophoresis) by adding ⁇ 32 P-dCTP (or any labeled nucleotide) to a 5 ⁇ l aliquot of the reaction mixture.
  • the RNA may be hydrolyzed by the addition of 2 ⁇ l of 0.5 M EDTA (pH 8.0) and 5 ⁇ l of 0.5 N NaOH, followed by incubation at 70° C for 1 hour. This reaction mixture can be brought to neutral pH by the addition of 5 ⁇ l of 1 M Tris-HCl (pH 7.5). The DNA can then be separated from the unincorporated dNTPs and RNA fragments by centrifuging the sample through an appropriate DNA purification column, such as a ChromaSpin-400 column (Clontech) .
  • an appropriate DNA purification column such as a ChromaSpin-400 column (Clontech) .
  • a DNA tail may be added to the first DNA strand in a reaction containing 50 ⁇ l of the first DNA strand from the above reaction, 14 ⁇ l of 5X TdT buffer (500 mM potassium cacodylate (pH 7.2), 110 mM cobalt chloride, and 1 mM DTT) , 3.5 ⁇ l of 1 mM of the preferred dNTP for tailing (e.g., dTTP), and 1.4 ⁇ l of TdT (15 U/ ⁇ l).
  • the reaction mixture is incubated at 37° C for 20 minutes before it is stopped by the addition of 2 ⁇ l of 0.5 M EDTA.
  • the tailed DNA then is separated from the unincorporated dNTPs by centrifuging the sample through a ChromaSpin-100 column, for example.
  • the tailed first strand should be present in a volume of approximately 80 to 90 ⁇ l.
  • the tailed first strand is mixed with 400 ng of the primer (e.g., a hybrid primer composed of 28 nucleotides of poly(rA) (the first oligonucleotide) bonded to 2 nucleotides which are degenerate in sequence (excluding dA at the 5' terminal position; the second oligonucleotide of the primer) ) .
  • the reaction also contains 20 ⁇ l of a conventional 10X DNA polymerase I buffer and 10 ⁇ l DNA polymerase I (10 U/ ⁇ l) in a final volume of 200 ⁇ l.
  • the reaction mixture is incubated at 16° C for 2.5 hours before 9 U of RNase H and 9 U of T4 DNA polymerase are added.
  • the reaction mixture then is incubated at 37° C for 30 minutes.
  • the synthesized DNA can be purified, e.g., on a ChromaSpin- 100 column, then precipitated with ethanol, and resuspended in the presence of 3.2 ⁇ g EcoRI adaptors, 1 mM ATP, and 4 U of T4 DNA ligase in a conventional ligase buffer in a total of 10 ⁇ l.
  • the .EcoRI adaptors are added by incubating this reaction mixture overnight at 4° C.
  • the adaptors may then be phosphorylated by adding to the sample 6 ⁇ l of H 2 0, 2 ⁇ l of 10 mM ATP, and 10 U of polynucleotide kinase in IX ligase buffer. This reaction mixture should be incubated at 37° C for 30 minutes.
  • the ss ends of the adaptors are then exposed by digesting the DNA at the Xhol site (e.g., by adding 120 U Xhol and 28 ⁇ l Xhol buffer supplement (Stratagene, La Jolla, CA) and incubating the sample at 37° C for 1.5 hours).
  • the ds DNA containing the adaptors can then be purified by centrifugation through a ChromaSpin-100 column, for example.
  • the DNA then is precipitated with ethanol and dissolved in 3 ⁇ l H 2 0, 0.5 ⁇ l of 10 mM ATP, 0.5 ⁇ l of IOX ligase buffer, 2 U of T4 DNA ligase, and a linear virus- based vector having appropriate cloning sites (e.g., 1.0 ⁇ l of the lambda-based ZAP Express Arms (Statagene) ) . After an overnight incubation at 12° C, the DNA may be packaged into phage.
  • a linear virus- based vector having appropriate cloning sites
  • Example III Preparation of Full-Length ds DNA From a DNA Template Using a Non-homopolymeric Tail
  • Fig. 7 An illustration of such a method is provided in Fig. 7.
  • the steps of the method shown in Fig. 7 are similar to the steps in Example I.
  • a tail which is non-homopolymeric in this case (5'prGrCrUrA3') , is added to the 3' end of the first DNA strand.
  • T4 RNA ligase may be used to attach a pre-formed tail to the DNA.
  • the first oligonucleotide of the primer which is annealed to the tailed DNA strand in step (b) is, of course, also non-homopolymeric.
  • the primer does not include the second oligonucleotide of the primer, which is optional because the first oligonucleotide of the primer is non-homopolymeric.
  • DNA polymerase is used to synthesize the second DNA strand in step (c) ; the strand and direction of synthesis are indicated by the arrow.
  • a ds RNAse is used to remove the tail and the first oligonucleotide of the primer in step (d) .
  • Example IV Preparation of Full-Length ds DNA Having a ss DNA Extension
  • the invention provides a convenient method for preparing ds DNA having a ss DNA extension to facilitate subsequent cloning of the DNA.
  • the method illustrated in Fig. 8 involves the following steps:
  • step (a) of Fig. 8 the 3' terminal dNTPs of the DNA strand are
  • the tail is generally attached to the 3' end of the DNA strand via ligation of a suitable pre-formed homopolymeric or non-homopolymeric oligonucleotide to the 3' end of the DNA strand, although sequential addition of nucleotides by an enzyme such as TdT or polyA polymerase would also suffice.
  • the non- homopolymeric tail is added by T4 RNA ligase.
  • the precise length of the tail is not critical, provided that it is long enough to hybridize efficiently and stably with the primer (i.e., at least four nucleotides long) . A range of approximately four to 50 nucleotides is recommended for the length of the tail. If the tail is to be the ss DNA extension, its length and sequence may be dictated by the length and sequence desired for the ss DNA extension. If one wishes to use a tail that is longer than the desired ss DNA extension, the length of the ss DNA extension can be modulated in the method by
  • step (b) A single-stranded primer complementary to all or a portion of the tail and a portion of the first DNA strand immediately adjacent to the 5' end of the tail is then allowed to hybridize with the tailed DNA strand.
  • the primer is complementary to the entire tail and two nucleotides of the first DNA strand.
  • the primer is conveniently thought of as consisting of two oligonucleotides, the first of which is complementary to a portion of the tail and which may consist of between four and 50 nucleotides (here, 5'dAdAdTdT3') .
  • This oligonucleotide of the primer can be either RNA or DNA.
  • the second oligonucleotide of the primer which is at least one nucleotide in length (and optional where a non-homopolymeric first oligonucleotide is used) , consists of DNA (here,
  • either (i) the tail or (ii) the first oligonucleotide of the primer, but not both, must be RNA.
  • the tail is RNA
  • the first oligonucleotide of the primer is therefore DNA.
  • the primer may be shorter than, longer than, or the same length as the tail.
  • the second DNA strand is synthesized by adding DNA polymerase and dNTPs to the primed template (Fig. 8; step (c) , with the synthesized strand being indicated by the arrow) .
  • RNA RNA
  • RNase H RNase H
  • the DNA portion of the tail or the first oligonucleotide of the primer will thereupon be rendered single-stranded.
  • This single-stranded extension of the ds DNA can be used as a "sticky end" to facilitate cloning of the DNA into a cloning vector (e.g., an expression vector) .
  • the method can be used, for example, to create a vectored cDNA library.
  • the sequence of the ss DNA extension is dictated by the sequence of the tail, which can be pre-determined by the practitioner. If desired, the ss DNA extension can be designed such that it is complementary (i.e., capable of hybridizing) to the sequence of a single- stranded extension produced by a restriction enzyme. In this example, the ss DNA extension is complementary to an .EcoRI sticky end. In general, where the restriction enzyme produces a 5' extension (e.g., as EcoRI does), the tail should include RNA having a sequence identical to the 5' extension (e.g., 5'rArArUrU3' for .EcoRI).
  • the tail should include DNA having the sequence of the 3 ' extension (e.g., 5'dTdGdCdA3' for PstI) .
  • the DNA having a ss extension produced in the method of the invention can readily be cloned into a vector which has been digested with the appropriate restriction enzyme (.EcoRI or PstI in the above examples) .
  • the sequence of the ss DNA extension and the sequence of the vector can be designed to place the promoter and ribosome binding sites of the vector immediately adjacent to the cDNA, thereby minimizing or eliminating the presence of unwanted sequences.
  • the invention can be used to produce a ds DNA molecule having an adaptor, which is a ds nucleic acid which has a ss extension.
  • the ss extension may be chosen by the practitioner, and it may be homopolymeric or non- homopolymeric.
  • the adaptor consists of DNA, although RNA may be used.
  • an ss DNA extension of an adaptor may be complementary to a ss DNA extension produced by a restriction enzyme, thereby increasing the efficiency with which the resulting DNA can be cloned.
  • the adaptor may be designed such that, when the DNA having the adaptor is ligated to a second DNA (e.g., a vector) which has been digested with a restriction enzyme, the resulting ligated DNA molecule contains a functional restriction enzyme recognition site formed by the adaptor and a portion of the second DNA.
  • a second DNA e.g., a vector
  • the resulting ligated DNA molecule contains a functional restriction enzyme recognition site formed by the adaptor and a portion of the second DNA.
  • one or a few base ⁇ pairs of the ds portion of the adaptor are identical to a portion of a restriction enzyme recognition site (the portion which remains double stranded after digestion) .
  • a DNA tail is added to the 3 ' end of a first DNA strand.
  • a DNA primer which produces an adaptor having a 3 ' or 5' ss DNA extension.
  • a primer which is shorter than the tail produces an adaptor having a 3' ss DNA extension (as is shown in Fig. 9, step (b) ) .
  • the primer may be designed such that a portion of it is complementary to the first DNA strand. Where a homopolymeric tail is used, it is preferable to use a primer which in part is complementary to a portion of the first DNA strand.
  • the adaptor is complementary to a "sticky end" produced by PstI.
  • the adaptor can readily be used to ligate the DNA molecule to a second molecule which has a PstI sticky end.
  • the resulting DNA molecule thus contains a complete PstI site (shown boxed) which subsequently can be cleaved by PstI (at the positions indicated by the arrowheads) .
  • this method of the invention offers flexibility in genetic manipulation techniques.
  • the DNA may be a cDNA, and a plurality of cDNAs may be used in the creation of a cDNA library.
  • the invention can be used to produce a ds DNA having a ds DNA protrusion.
  • a tail is first added to the 3 ' end of the first DNA strand (step (a)).
  • This method is a modification of that described in Example V in that the primer and tail do not create a ss DNA extension, but rather are designed such that they create a flush end, when annealed.
  • the primer and the tail are the same length or the primer is shorter than the tail, provided that they create a flush end.
  • the primer and tail should nonetheless be designed such that the 5* * end of the primer creates a flush end with the 3' end of the tail, as in Fig. 10, step (b) .
  • the primer in the presence of dNTPs and DNA polymerase, primes synthesis of the second DNA strand (step (c) ) .
  • synthesis employs methylated dATP in lieu of dATP, and the synthesized DNA is resistant to cleavage by EcoRI.
  • the ds DNA having a ds protrusion may be cloned, e.g., by blunt-end ligation, into a vector.
  • the tail and primer may be selected such that the DNA protrusion has a particular, desirable sequence.
  • the protrusion may include a promoter and ribosome binding site to direct expression of the downstream DNA in a prokaryote.
  • the DNA protrusion may be designed to contain a restriction enzyme recognition site; in the illustrated example, the DNA protrusion contains an EcoRI site (shown boxed, with the sites of cleavage marked by the arrowheads) .
  • the resulting DNA molecule having a ds protrusion may be cloned by blunt- end ligation into a cloning vector, creating a DNA construct which contains a restriction enzyme site useful for subsequent genetic manipulation.
  • the ds DNA molecule having a ds protrusion may be digested with a restriction enzyme prior to inserting the DNA into a cloning vector (Fig. 10, step (d) ) .
  • a restriction enzyme prior to inserting the DNA into a cloning vector
  • the additional nucleotides facilitate binding of the restriction enzyme to the DNA molecule, increasing the efficiency with which the restriction enzyme cleaves the DNA molecule.
  • the "sticky ends" facilitate subsequent cloning of the DNA.
  • Example VII PCR Amplification of ds DNA
  • the methods described in Examples I, III, IV (where the primer includes RNA) , and VI can be adapted to permit amplification of a sample of ds DNA (such as ds cDNA) by PCR, as follows: to a sample of ds DNA prepared by a method of the invention, with its primer/tail extension still intact, is added excess primer (identical to the primer used to generate the original ds DNA) and excess oligo(dT) primer, in a reaction mixture with an appropriate temperature-stable DNA polymerase (e.g., Taq) and dNTPs; the mixture is subjected to an appropriate number of PCR temperature cycles in a PCR machine (e.g., 40 cycles) in accordance with standard PCR procedures.
  • an appropriate temperature-stable DNA polymerase e.g., Taq
  • the primer/tail extensions on each ds DNA molecule so generated can be removed, as described above.
  • RNA strand such as an mRNA strand or a viral RNA.
  • a homopolymeric or non-homopolymeric RNA tail is then added, preferably by ligation, to the 3' end of the RNA, and a homopolymeric or non-homopolymeric RNA leader is, simultaneously or sequentially, added to the 5' end of the RNA.
  • RNA leader is added to mRNA, conventional methods can be used to remove the 7- methylguanosine cap structure typically present on eukaryotic mRNA.
  • tobacco acid pyrophosphatase and bacterial acid phosphatase can be used to remove the cap and 5' phosphates.
  • the mRNA can subsequently be phosphorylated with T4 polynucleotide kinase to produce a mRNA having a single phosphate group at its 5' end.
  • TAP tobacco acid pyrophosphatase
  • bacterial acid phosphatase can be used to remove the cap and 5' phosphates.
  • the mRNA can subsequently be phosphorylated with T4 polynucleotide kinase to produce a mRNA having a single phosphate group at its 5' end.
  • TAP tobacco acid pyrophosphatase
  • bacterial acid phosphatase can be used to remove the cap and 5' phosphates.
  • the mRNA can subsequently be phosphorylated with T4 polynucleotide kinase to produce a mRNA having a single phosphate group at
  • An appropriate reaction mixture for attaching the 5' leader to the RNA includes, for example, 5 ⁇ g RNA, 10 units T4 RNA ligase, and 20 mM leader.
  • the RNA tail can be added according to procedures described for other methods of the invention. As in other methods of the invention, the sequence of the tail and, in this case, the leader as well, may be pre-determined by the practitioner. In the illustrated example, the RNA leader lacks a 5*" phosphate, preventing it from being ligated to the 3' end of the RNA, whereas the RNA tail has a 5' phosphate (step (a) ) . Where the RNA tail and leader differ in sequence, it is best to add them sequentially (e.g., by exploiting the requirement for a 5' phosphate).
  • RNA primer is then hybridized to all or a portion of the RNA tail. If desired, this RNA primer may be complementary to one to five nucleotides at the 5' end of the RNA, as well as to the RNA tail. Such an "overlapping" primer is preferred if the RNA tail is a homopolymer, since it serves in such a case to position the primer correctly.
  • the first RNA primer hybridizes to all of the RNA tail but not the non-tail RNA. This first primer is used to initiate synthesis of the first DNA strand in the presence of DNA polymerase and dNTPs, using the RNA having the tail and leader as a template (step (c) ) .
  • Denaturation (i.e., separation of the strands) of the resulting ds nucleic acid provides a ss DNA/RNA template for synthesis of a second DNA strand (step (d)) .
  • This second strand is primed by a RNA primer which hybridizes to the 3* * end of the first DNA strand.
  • This second primer may be complementary to all or a portion of the region of the first DNA which is complementary to the RNA leader. Accordingly, the second RNA primer may be identical in sequence to all or a portion of the RNA leader.
  • the second primer used in step (e) of the example is identical to the leader and complementary to a portion of the 3' end of the first DNA strand.
  • Synthesis of the second DNA strand results in a ds nucleic acid having the first RNA primer at one 5' end and the second RNA primer of the other 5' end.
  • Removal of the RNA portions of the ds nucleic acid results in a ds DNA having two ss DNA extensions.
  • the sequences of these extensions may be dictated by the sequences of the tail and leader.
  • the ss DNA extensions on the ds DNA may be used to facilitate cloning of the DNA into an expression vector, for example. For cloning the DNA, it may be desirable to design the ss DNA extensions so that they are complementary to ss DNA extensions produced by restriction enzymes.
  • RNA tail having the sequence 5'rUrGrCrA3' and a complementary first RNA primer having the sequence 5'rUrGrCrA3'
  • a ss DNA extension which has the sequence 5 'dTdGdCdA3', which is complementary to a ss DNA extension produced by PstI (i.e., able to hybridize to a PstI "sticky end" under standard conditions for DNA cloning) .
  • RNA leader having the sequence 5'rCrArUrG3' and a second RNA primer having the sequence 5'rCrArUrG3'
  • ss DNA extension having the sequence 5 'dCdAdTdG3' , which is complementary to a ss DNA extension produced by SphI .
  • Example IX Comparison of the Compositions of Two Populations of RNA Molecules Described in this example are convenient methods for comparing the compositions of two populations of RNA molecules.
  • the method illustrated in Figs. 12a and 12b involves the following steps:
  • RNA molecules e.g., mRNA
  • the DNA strands in the first population will have different and probably random sequences at their 3*" ends; the three different DNA molecules are labeled I, II, and III throughout the figure.
  • a homopolymeric tail (as is shown in Fig. 12a step (a)) or non-homopolymeric tail of at least four nucleotides is then added to the 3' end of each strand in the first population of DNA strands, to yield a first population of tailed DNA strands.
  • the homopolymeric tail is added using dCTP and TdT.
  • a first set of ss primers is then allowed to hybridize to the tails of the tailed DNA strands.
  • Each primer in the first set consists of two portions referred to as a "first oligonucleotide” and a “second oligonucleotide,” respectively.
  • the second oligonucleotide is optional where a homopolymeric tail is used.
  • the first oligonucleotide is at least four nucleotides complementary to all or a portion of the tail, and the second oligonucleotide, which is covalently bonded to the 3* * terminus of the first oligonucleotide, consists of one or more deoxyribonucleotides.
  • the sequence of the second oligonucleotide is fully degenerate within the first set of primers, with one exception: where the first oligonucleotide is a homopolymer, at least one nucleotide of the second oligonucleotide is not identical to the nucleotide which composes the homopolymer. This non- identical nucleotide is preferably the 5' terminal nucleotide of the second oligonucleotide.
  • the 5' terminal nucleotide is specified as being dA, dC, or dT, none of which is identical to nucleotide of the homopolymer (dG) .
  • the set of primers is designed to contain a primer having a second oligonucleotide complementary to each of the DNA strands present in the first population of DNAs so that every DNA strand in the first population of DNAs will be able to hybridize to a primer from the first set of primers.
  • the first oligonucleotide and tail need not be removed in this method, the first oligonucleotide and the tail may both be RNA or DNA, or one may be RNA while the other is DNA.
  • the tail is DNA and the first oligonucleotide of the primer is RNA.
  • the primer may be shorter than, longer than, or the same length as the tail.
  • a second set of ss primers is also added (step (c) ) .
  • the second set of primers consists of oligonucleotides which may all have the same sequence, or which may represent a plurality of sequences. The sequence(s) will typically be randomly selected. Preferably, these primers are relatively short in length (e.g., seven to 15, preferably eight to 12, or more preferably nine to 11 nucleotides) .
  • step (d) Once the first set of primers is annealed, primer-dependent synthesis of the sense strand ensues in the presence of DNA polymerase and dNTPs, yielding a first population of ds DNAs (step (d) ) . After denaturing this ds DNA, both the first and the second sets of primers are used to prime synthesis of multiple copies of ds DNA (the synthesized strands are indicated with arrows (Fig. 12b)). Each parental molecule (I, II, and III) gives rise to two synthesized molecules (designated I-A, I-B, II-A, II-B, III-A, and III-B) .
  • the second set of primers is designed to have one or a few sequences which are complementary to randomly located sites on a random subset of the second DNA strands, so that amplification (e.g., by PCR) of a given RNA population with the two sets of primers yields an assortment of fragments of varying lengths that is characteristic of that population. Because the second set of primers has sequences which anneal at varying positions along the DNA templates, ds molecules synthesized from them are "short products" of varying lengths (i.e., shorter than the original DNA molecules) .
  • PCR can be used to amplify the copy number of the characteristic set of ds DNAs, so that the first population of ds DNAs can be subjected to further analysis.
  • the ds DNAs can be separated by size, e.g., with high resolution polyacrylamide gel electrophoresis, to produce characteristic patterns of bands corresponding to the components of the population.
  • steps (a) through (d) are also performed for the second population of first DNA strands, to produce a second population of ds DNAs which has a size distribution that is characteristic of the second population of RNAs. Constitutive differences between the two populations of RNA molecules will be indicated by differences in the characteristic patterns produced.
  • Example X Identification of a Gene Which is Differentially Expressed Between Populations of Cells
  • the above method may be adapted for the purpose of identifying a gene which is expressed differentially between populations of cells: i.e., which is more highly expressed in one type of cell than in another or expressed in one cell type but not in another.
  • particularly interesting genes are those which are differentially expressed (i) during development, (ii) as part of the etiology of cancer, or (iii) in cells infected with a pathogen (versus uninfected cells) .
  • This method is a variation of the method of Example IX for comparing the compositions of populations of RNAs.
  • a first population of cDNA strands which are complementary to mRNA species of a first population of cells, e.g., cells derived from a breast tumor.
  • the cDNA strands can be synthesized with conventional methods.
  • tails are then added to the cDNA strands, primers are annealed to the tails and the ds DNA is synthesized, with synthesis being primed from primers of both sets of primers.
  • the tail and the "first oligonucleotide" portion of the primer may be removed, if desired.
  • the procedure is repeated on a second population of DNAs derived from a second cell type - e.g., normal breast cells.
  • Conventional methods e.g., high-resolution polyacrylamide gel electrophoresis
  • a DNA molecule which is present in one of the populations of DNA molecules and which is absent or present at a reduced level in the other population of molecules represents a gene which is differentially expressed between the cell types.
  • Previously-described techniques can then be used to isolate and clone the DNA of the differentially expressed gene. This method is conceptually similar to Liang and Pardee's differential display technique (Science 257:967-971, 1992), but provides a means to focus on the 5' end of mRNAs rather than the 3 ' end.
  • the tail can be added to the first DNA strand while the latter is part of a ds nucleic acid, e.g., a mRNA-cDNA hybrid.
  • the second nucleic acid strand e.g., the mRNA
  • the nucleotides which compose the tail and the primer are not limited to A, C, G, T, and U.
  • Modified nucleotides e.g., inosine, methylated dATP, or methylated dCTP
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGAATTCAT 10

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Abstract

Des procédés, comme celui présenté dans la figure, permettent d'obtenir un ADN double-brin à partir d'une matrice d'ADN linéaire. Ils consistent 1) à ajouter une queue en 3' à un brin d'ADN, 2) à hybrider une amorce avec cet ADN doté d'une queue, amorce complémentaire de la queue en 3' et d'une partie de l'ADN, et 3) à synthétiser un deuxièeme brin d'ADN. On peut utiliser ces procédés pour produire des molécules d'ADN double-brin à extrémités franches ou des molécules d'ADN à prolongements d'ADN simple-brin qui facilitent le clonage ultérieur de l'ADN. On décrit aussi des procédés permettant de comparer les compositions de deux populations de molécules d'ARN et d'identifier un gène exprimé de façon différentielle dans deux types de cellules.
PCT/US1996/014046 1995-08-28 1996-08-26 Utilisation d'une amorce de positionnement en 5' pour produire un adn double-brin a partir d'une matrice d'adn WO1997008185A1 (fr)

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AU69103/96A AU6910396A (en) 1995-08-28 1996-08-26 Use of a 5' positional primer to produce double-stranded dna from a dna template

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
FR2783000A1 (fr) * 1998-09-04 2000-03-10 Genolife Procede d'amplification de la totalite des fragments d'adn d'un echantillon
WO2001071027A2 (fr) * 2000-03-24 2001-09-27 Micromet Ag Amplification d'arn messager
US6358712B1 (en) * 1999-01-05 2002-03-19 Trustee Of Boston University Ordered gene assembly
JP2002537762A (ja) * 1999-01-05 2002-11-12 トラスティーズ オブ ボストン ユニバーシティ 改良型核酸クローニング
US7432084B2 (en) * 2001-08-31 2008-10-07 Rosetta Inpharmatics Llc Methods for preparing nucleic acid samples

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US5162209A (en) * 1991-01-18 1992-11-10 Beth Israel Hospital Association Synthesis of full-length, double-stranded dna from a single-stranded linear dna template
US5262311A (en) * 1992-03-11 1993-11-16 Dana-Farber Cancer Institute, Inc. Methods to clone polyA mRNA

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US5162209A (en) * 1991-01-18 1992-11-10 Beth Israel Hospital Association Synthesis of full-length, double-stranded dna from a single-stranded linear dna template
US5262311A (en) * 1992-03-11 1993-11-16 Dana-Farber Cancer Institute, Inc. Methods to clone polyA mRNA

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014277A1 (fr) * 1998-09-04 2000-03-16 Genolife Procede d'amplification de la totalite des fragments d'adn d'un echantillon
US6605435B1 (en) 1998-09-04 2003-08-12 Genolife Method for amplifying all of the DNA fragments in a sample, including small and damaged fragments, by pretreating the fragments with P1 nuclease
FR2783000A1 (fr) * 1998-09-04 2000-03-10 Genolife Procede d'amplification de la totalite des fragments d'adn d'un echantillon
AU774306B2 (en) * 1999-01-05 2004-06-24 Trustees Of Boston University Improved nucleic acid cloning
US7439021B2 (en) * 1999-01-05 2008-10-21 Trustees Of Boston University Nucleic acid cloning
US6358712B1 (en) * 1999-01-05 2002-03-19 Trustee Of Boston University Ordered gene assembly
JP2002537762A (ja) * 1999-01-05 2002-11-12 トラスティーズ オブ ボストン ユニバーシティ 改良型核酸クローニング
WO2001071027A3 (fr) * 2000-03-24 2003-10-09 Micromet Ag Amplification d'arn messager
EP1728874A1 (fr) * 2000-03-24 2006-12-06 Micromet AG Méhode pour la préparation d' un surrogate in vitro pour identifier les gènes exprimés dans un échantillon test
US7229760B2 (en) 2000-03-24 2007-06-12 Micromet Ag mRNA amplification
AU2001242509B2 (en) * 2000-03-24 2007-08-23 Micromet Ag mRNA amplification
WO2001071027A2 (fr) * 2000-03-24 2001-09-27 Micromet Ag Amplification d'arn messager
US7432084B2 (en) * 2001-08-31 2008-10-07 Rosetta Inpharmatics Llc Methods for preparing nucleic acid samples

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