WO2001083696A2 - PROCEDES D'ISOLEMENT ET DE SEQUENCAGE RAPIDES DE SEQUENCES GENETIQUES SPECIFIQUES - Google Patents

PROCEDES D'ISOLEMENT ET DE SEQUENCAGE RAPIDES DE SEQUENCES GENETIQUES SPECIFIQUES Download PDF

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WO2001083696A2
WO2001083696A2 PCT/US2001/013807 US0113807W WO0183696A2 WO 2001083696 A2 WO2001083696 A2 WO 2001083696A2 US 0113807 W US0113807 W US 0113807W WO 0183696 A2 WO0183696 A2 WO 0183696A2
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gene
primer
specific
sequence
seq
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PCT/US2001/013807
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English (en)
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Inc. Digital Gene Technologies
Rolf Muller
Gretchen H. Riddle
James R. Glass
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Digital Gene Tech Inc
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Priority to AU57411/01A priority Critical patent/AU5741101A/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
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a method for the rapid isolation and ' sequencing of novel gene-specific sequences. Additionally, the present invention relates to novel oligonucleotide primer sequences and compositions thereof and kits comprising such oligonucleotide primer sequences and compositions.
  • Nucleotide sequence fragments of unknown genes can be obtained by various molecular biology techniques including protein sequencing, cDNA library screening, EST sequencing, and cloning of DNA fragments generated using jjolymerase chain reaction (PCR).
  • PCR jjolymerase chain reaction
  • some information regarding the structure and function of the gene may be inferred from the nucleotide sequence of the gene fragment.
  • the challenge is to isolate a single particular sequence extension from within a large mixture of other unwanted sequences.
  • PCR is a powerful technique for amplifying specific nucleic acid sequences (U.S. Pat No. 4,683, 202; U.S. Pat No. 4,683, 195; Erlich, PCR Technology, (Stockton Press, 1989)) and is becoming more widely used as a basis for isolating gene sequences and their characterization.
  • RACE Rapid Amplification of cDNA Ends
  • RNA ligase to add a known sequence to the end of the mRNA (Liu, X., and Gorovsky, M.A. (1993) Nucleic Acids Research, .21: 4954-60 (1993).
  • Other methods have added known sequences to the 5' end of cDNA libraries by using adapters or adapter like molecules (Chenchik et al., BioTech., 21 :526-534 (1996) and Tavtigian et al. (U.S. Pat.No. 5,789,206) and the resulting cDNA population is used as a PCR amplification template.
  • the 3'-RACE technique uses an adapter primer containing a unique sequence and polydT residues to reverse transcribe an mRNA population, producing a first strand cDNA.
  • a gene-specific primer is annealed with the first strand cDNA and extended to generate a complementary second strand.
  • the target cDNA is amplified using a gene-specific primer and the described adapter primer.
  • a gene-specific primer is used to reverse transcribe the mRNA, after which a poly(dA) tail (or other polydeoxynucleotide tail) is added to the primer extended product.
  • the second strand synthesis is carried out by annealing the adapter poly(dT) primer with the poly(dA) tail.
  • the target cDNA is amplified using the gene- specific primer and the adapter primer.
  • the need of cloning and clonal selection using screening methods like PCR or hybridization prior to sequence determination does not allow the method to be introduced in a robotics high-throughput environment needed in our genomics age.
  • the method described herein allows direct sequencing and identification of gene specific sequences and provide a description for a fully automatable process for obtaining extended and full-length gene sequences.
  • the present invention provides novel methods and composition for isolating and sequencing gene-specific polynucleotides.
  • the present invention provides several advantages over the available methods.
  • the elimination of cloning and screening clones provides quicker sequence results.
  • the capture of the gene-specific first strand significantly reduces the background of non-specific PCR products.
  • Amplification of the entire adapted cDNA template increases the ability to detect and sequence rare transcripts.
  • the method of the present invention is adaptable to multiwell formats, providing a high-throughput system for generation of extended and full-length sequences.
  • the method of the present invention is adaptable to automation using techniques of robotics, fluid handling and numeric control known to the skilled artisan, further increasing the throughput of a system for generation of extended and full-length sequences.
  • the present invention provides a novel method for isolating a gene-specific polynucleotide, which method comprises the steps of: (a) synthesizing a population of double stranded DNA molecules, wherein each strand has a 5' end and a 3' end, using an anchor primer having a first captureable moiety, wherein the anchor primer is at the 3' end of each double stranded DNA molecule; (b) ligating a double stranded adapter rare sequence molecule to the 5' end of each double stranded DNA molecule; (c) synthesizing a single stranded gene-specific polynucleotide using a gene-specific primer having a second captureable moiety, wherein the captureable moiety of the gene-specific primer is different from the captureable moiety of the anchor primer; (d) purifying the single stranded gene-specific polynucleotide; and (e) amplifying the purified gene-specific polynucleotide using both a gene-
  • the method comprises the additional step of sequencing the amplified isolated gene-specific polynucleotide.
  • the anchor primer comprises the sequence 5'- N'(N) a (T) b -3' where N' is a nucleotide to which a captureable moiety is attached, a is an integer of from about 15 to about 50, and b is an integer of from about 12 to about 25.
  • N' is a biotinylated nucleotide.
  • N' is an aminated nucleotide.
  • the anchor primer comprises the sequence 5'- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3* where N is a biotinylated guanidylate nucleotide (SEQ ID NO: 1).
  • the anchor primer comprises the sequence 5*- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3' where N is an aminated guanidylate nucleotide (SEQ ID NO: 2).
  • the double-stranded adapter molecule comprises the sequence of at least one rare restriction enzyme site wherein the rare restriction enzyme site occurs in a mammalian genome less than of about one site per 15,000 nucleotides.
  • the double-stranded adapter molecule comprises the sequence of two or more rare restriction enzyme sites which are sequentially arranged, selected from the group consisting of Nptf, Ascl, Bael, Fsel, Sfil, Sgfl, SerAI. Srfl. Pad. Pmel. PpuMI, RsrII. Sapl. SexAL Sse8387I. Sbfl. SpeL Sail. Rsrl, and Nhel restriction enzyme sites.
  • the double-stranded adapter molecule comprises the sequence of contiguous Sbfl. SpeL Sail. Rsrl. and Nhel restriction enzyme sites.
  • the double-stranded adapter molecule comprises an RNA polymerase promoter sequence selected from the group consisting of T3, SP6, and T7 RNA polymerase promoter sequences.
  • the double-stranded adapter molecule comprises the T7 polymerase promoter sequence.
  • the double-stranded adapter molecule comprises the sequence of 5'-AT- AGCCTGCAGGTAATACGACTCACTATAGGGACTAGTCGACGGACCGCTAG CATCAGATC - 3' (SEQ ID NO: 13).
  • the double stranded adapter molecule comprises SEQ ID NO: 15.
  • method comprises the step of amplifying the double stranded DNA molecules comprising both an anchor primer and an adapter molecule before the step of synthesizing the single stranded gene-specific polynucleotide, wherein the double stranded DNA molecules are amplified using a 3' end primer that hybridizes to a sequence located in the anchor primer and a 5' end primer that hybridizes to a sequence located in the adapter molecule.
  • the 3' end amplification primer comprises the sequence 5'- NAATTCAACTGGAAGCGGCCGCAGGA -3' (SEQ ID NO: 20 or 21) and the 5' end amplification primer comprises the sequence of 5' - CCTGCAGGTAATACGACTCACTATAGG- 3' (SEQ ID NO: 17). In one embodiment, the 5' end amplification primer comprises the sequence of SEQ ID NO: 18.
  • the method comprises the additional steps of: synthesizing a population of single-stranded RNA molecules from the population of double stranded DNA molecules comprising an anchor primer and an adapter molecule, and digesting the double stranded DNA template molecules, before the step of synthesizing the single stranded gene-specific polynucleotide.
  • the gene specific primer comprises the sequence 5'- N'(N) C -3' where N' is a nucleotide to which a captureable moiety is attached and c is an integer of from about 17 to about 40.
  • N' is a biotinylated nucleotide.
  • N' is an aminated nucleotide.
  • the first captureable moiety of the anchor primer of the double stranded DNA molecule is affixed to a substrate comprising a coating of streptavidin.
  • the first captureable moiety of the anchor primer of the double stranded DNA molecule is affixed to a substrate comprising a coating of N-oxysuccinimide ester.
  • the second captureable moiety of the gene-specific primer of the gene-specific single stranded polynucleotide is affixed to a substrate comprising a coating of streptavidin. In another embodiment, the second captureable moiety of the gene-specific primer of the gene-specific single stranded polynucleotide is affixed to a substrate comprising a coating of N-oxysuccinimide ester.
  • the purified single stranded gene-specific polynucleotide is amplified using a 5' end gene-specific primer and a 3 'end amplification primer, the 3 'end amplification primer hybridizing to a portion of the sequence of the anchor primer.
  • the 3' end amplification primer comprises SEQ ID NO: 23.
  • the 5' end amplification primer comprises SEQ ID NO: 17. In another embodiment, the 5' end amplification primer comprises SEQ ID NO: 18.
  • the present invention provides a method for isolating a gene-specific polynucleotide comprising the steps of: synthesizing a population of double stranded DNA molecules having a 3' end and a 5' end; ligating a double stranded adapter molecule to the 5' end of each double stranded DNA molecule; attaching at least one gene-specific oligonucleotide primer having a captureable moiety to a solid substrate, wherein the gene-specific primer is attached via the captureable moiety; synthesizing at least one single stranded gene-specific polynucleotide using the attached gene-specific primer(s); purifying the single-stranded gene-specific polynucleotide(s); and amplifying the gene-specific polynucleotide(s) using a gene-specific primer and an primer that hybridizes to a sequence located in the adapter molecule.
  • the second captureable moiety of the gene-specific primer of the gene-specific single stranded polynucleotide is affixed to a substrate comprising a coating of streptavidin. In another embodiment, the second captureable moiety of the gene-specific primer of the gene-specific single stranded polynucleotide is affixed to a substrate comprising a coating of N-oxysuccinimide ester.
  • the purified single stranded gene-specific polynucleotide is amplified using a 3' end gene-specific primer and a 5' end amplification primer that hybridizes to a sequence located in the adapter molecule.
  • the 5' end amplification primer comprises SEQ ID NO: 17.
  • the 5' end amplification primer comprises SEQ ID NO: 18.
  • the invention also provides an isolated polynucleotide having an attached captureable moiety chosen from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:45 and SEQ ID NO:46.
  • the invention provides an isolated double stranded polynucleotide chosen from the group consisting of SEQ ID NO: 13 and complementary sequence SEQ ID NO: 14, and SEQ ID NO: 15 and complementary sequence SEQ ID NO: 16.
  • the invention provides an isolated polynucleotide comprising the sequence chosen from the group consisting of SEQ ID NO: 17-19.
  • the invention also provides an isolated polynucleotide comprising an anchor amplification primer oligonucleotide having an attached biotin moiety.
  • the anchor primer is an isolated polynucleotide selected from the group consisting of:
  • the anchor amplification primer oligonucleotide has an attached primary amine comprising the sequence consisting of SEQ ID NO: 21.
  • the anchor amplification primer oligonucleotide comprises the sequence consisting of SEQ ID NO: 23.
  • the invention provides a composition comprising the oligonucleotide and an aqueous carrier.
  • the invention provides an article of manufacture comprising the oligonucleotide and a streptavidin-coated substrate.
  • the invention provides a kit suitable for isolating and sequencing a gene-specific polynucleotide from a population of DNA or RNA polynucleotides comprising at least one single stranded anchor primer oligonucleotide having an attached captureable moiety and at least one double stranded adapter oligonucleotide in an amount sufficient for at least one assay and suitable containers.
  • the kit further comprises at least one single stranded amplification anchor primer oligonucleotide and at least one single stranded amplification adapter primer oligonucleotide.
  • the kit further comprises at least one single stranded gene-specific primer oligonucleotide having a captureable moiety, wherein the captureable moiety of the anchor primer oligonucleotide is different from the captureable moiety of the gene-specific primer oligonucleotide.
  • the kit typically comprises the gene-specific primer oligonucleotide is attached to a substrate comprising a coating. In one embodiment, the substrate is a multiwell plate.
  • the present invention provides a method for determining without cloning an extended sequence of a gene-specific polynucleotide that has a partial gene-specific sequence known, comprising the steps of: synthesizing a population of double stranded DNA molecules having a 3' end and a 5' end using an anchor primer having a first captureable moiety, wherein the anchor primer is at the 3' end of each double stranded DNA molecule; ligating a double stranded adapter molecule to the 5' end of each double stranded DNA molecule producing double stranded DNA molecules comprising both an anchor primer and an adapter molecule; synthesizing a single stranded gene-specific polynucleotide using a gene- specific oligonucleotide primer having a second captureable moiety, wherein the captureable moiety of the gene-specific oligonucleotide primer is different from the captureable moiety of the anchor primer; isolating the single stranded gene-
  • the present invention provides a method for the simultaneous isolation and sequence determination of multiple novel genes, comprising the steps of: (a) synthesizing a population of double stranded DNA molecules, wherein each strand has a 5' end and a 3' end; (b) ligating a double stranded adapter molecule to the 5' end of each double stranded DNA molecule; (c) attaching at least one gene-specific oligonucleotide primer having a captureable moiety to a solid substrate, wherein the gene-specific primer is attached via the captureable moiety; (d) synthesizing at least one single stranded gene-specific polynucleotide using the attached gene-specific primer(s)and a double stranded DNA having a 5' end adapter as a template; (e) purifying the single stranded gene-specific polynucleotide(s); and (f) amplifying the gene-specific polynucleotide(s) using both a gene
  • the present invention provides novel oligonucleotide primer sequences which can be used to practice preferred embodiments of the present invention. Also provided are compositions which comprise a novel oligonucleotide primer and an aqueous carrier. The present invention also provides kits for the rapid isolation and sequencing of novel genes from DNA and RNA libraries which comprise novel oligonucleotide primers.
  • Figure 1 shows the production of a 3' end capture modified cDNA (cDNA M ) using a captureable anchor primer.
  • FIG. 2 shows the production of a specially adapted cDNA (acDNA M ) by attachment of a double stranded adapter molecule to the 5' end of the cDNA M .
  • Figure 3 shows the production of a gene-specific sequence oriented in either the mRNA sense direction or the mRNA antisense direction.
  • Figure 4 summarizes anchor amplification primers and adapter amplification primers used.
  • Figure 5 shows the purification of a gene-specific sequence from a biotinylated cDNA library using aminated capture.
  • Figure 6 shows the purification of a gene-specific sequence from an aminated cDNA library using biotinylated capture.
  • Figure 7 shows the purification of a gene-specific sequence from a synthetic RNA library.
  • Figure 8 is a schematic diagram showing the adapter and gene-specific primers as well as the sequencing strategy used to determine the sequence of the cyclophilin gene.
  • Figure 9 is a schematic diagram showing the adapter and gene-specific primers as well as the sequencing strategy and primer walking used to determine the sequence ofa novel M41 gene.
  • Figure 10 is a schematic diagram showing the adapter and gene-specific primers as well as the sequencing strategy and primer walking used to determine the sequence of a novel M51 gene.
  • the orientation of a nucleic acid is described in terms of reference to the mRNA ("sense" RNA).
  • sense RNA
  • reference to the 5' end and 3' end of a double stranded DNA is meant to describe those ends corresponding to the 5' end and 3' end, respectively, of the mRNA.
  • a primer that hybridizes to the 3' end of a double stranded DNA is oriented in the 5' or antisense direction.
  • a primer that hybridizes to the 5' end of a double stranded DNA is directed in the 3' or sense direction.
  • polynucleotide is defined to encompass DNA and RNA of both synthetic and natural origin.
  • the polynucleotide may exist as single or double stranded DNA or RNA, or an RNA/DNA heteroduplex.
  • the polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified" bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
  • oligonucleotide refers to a single stranded DNA or
  • RNA molecule refers to an oligonucleotide that is hybridized to a nucleic acid template for sequencing purposes or to prime enzymatic synthesis of a second nucleic acid strand.
  • an "oligonucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO: 1-50.
  • An “oligonucleotide” of the present invention also includes those oligonucleotides capable of hybridizing, under stringent hybridization conditions, to the complementary sequences contained in SEQ ID NO: 1-50.
  • “Stringent hybridization conditions” refers to an overnight incubation at 42° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing in O.lx SSC at about 65°C.
  • An "oligonucleotide” of the present invention also includes those oligonucleotides that are substantially identical to the oligonucleotide sequences of SEQ ID NO: 1-50.
  • substantially identical refers to a nucleotide sequence that is at least about 80% identical to the reference oligonucleotide. More preferably, a "substantially identical" sequence is at least about 90% identical, and most preferably at least about 95-99% identical to a sequence contained in SEQ ID NO: 1-50.
  • identity is well-known to skilled artisans (Carillo, et al., S M J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Bishop, M.J., Ed., Guide to Huge Computers, (Academic Press, San Diego, (1994)), and Carillo (1988), supra. Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux, et al., Nuc. Acids Res.
  • the parameters are set so that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). The result of said global sequence alignment is in percent identity.
  • the term "double stranded” refers to a pair of nucleotide molecules that exist in a hydrogen-bonded arrangement typically assiated with DNA. Included are paired polynucleotides and oligonucleotides that are essentially double stranded, meaning that they may contain short regions of mismatch, such as from one to about three nucleotides, resulting from design or error.
  • the term "substantially complementary” refers to the hybridization or base pairing between nucleotides, such as, for example, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleotide to be amplified or sequenced.
  • Two single stranded nucleotide molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned with appropriate nucleotide insertions, deletions or substitutions, pair with at least about 80% of the nucleotides of the other strand.
  • the "captureable anchor primer” as that term is used herein refers to the modified polydT primer used to generate the second strand nucleic acid from the template strand nucleic acid.
  • the captureable anchor primer comprises a single stranded oligonucleotide sequence wherein a polydT sequence is found at the 3' end and a captureable moiety is found at the 5' end of the oligonucleotide.
  • captureable moiety refers to any functional group or molecule that can be joined to a nucleotide and can be attached to a substrate or solid- phase support.
  • the captureable moiety can be directly or indirectly attached to a nucleotide and can also be directly or indirectly attached to a subsfrate or solid-phase support.
  • spacer is intended to encompass any suitable means that can be used to link a nucleotide of the anchor primer to the captureable moiety or any suitable means by which to link the captureable moiety to the solid-phase support. The spacer should not adversely affect the function of the anchor primer or the captureable moiety.
  • the term "substrate” or “solid-phase support” is defined as any material having a rigid or semi-rigid surface.
  • the substrate or solid support should have a surface chemistry that allows the substrate to form a direct attachment with a captureable moiety.
  • the substrate or solid support should have a surface that can be coated or derivatized such that, upon coating or derivatization, the substrate can form an attachment with a captureable moiety.
  • the "adapter molecule” as that term is used herein refers to the double stranded polynucleotide which is ligated to the 5' end of each cDNA M having a 3' end modified with a captureable anchor primer, thereby generating an adapted cDNA M (acDNA M ).
  • rare restriction enzyme site refers to a restriction enzyme site that has at least six bases and occurs in a mammalian genome with a maximum frequency of about one site per 10,000 nucleotides.
  • RNA polymerase promoter site refers to the consensus sequence of the promoter site to which an RNA polymerase binds to initiate RNA synthesis.
  • the “anchor amplification primer” (An) refers to the 3' end oligonucleotide of an oligonucleotide primer pair used to amplify the acDNA M library.
  • the An primer hybridizes with a sense anchor primer sequence for the synthesis of a product extended in the 5' or antisense orientation.
  • the "adapter amplification primer”(Ap) refers to the 5' end oligonucleotide of an oligonucleotide primer pair used to amplify the acDNA M library.
  • the Ap primer hybridizes with an antisense adapter molecule sequence for the synthesis of a product extended in the 3' or sense orientation.
  • the "captureable gene-specific primer” as that term is used herein refers to the oligonucleotide primer used to generate the first strand nucleic acid of a gene-specific sequence.
  • the captureable gene-specific primer comprises a single stranded oligonucleotide sequence wherein a captureable moiety is attached to the 5' end of the oligonucleotide.
  • the "gene-specific amplification primer” refers to the oligonucleotide primer used to amplify isolated gene-specific cDNA.
  • the Gs primer is selected to be fully or substantially complementary to a known gene-specific sequence on the sense or antisense strand of a purified gene-specific cDNA.
  • the Gs primer may comprise the same nucleotide sequence as the gene-specific primer used to generate the first strand of a gene-specific cDNA.
  • nested oligonucleotide primer refers to an oligonucleotide primer that hybridizes with an internal sequence in the first-reaction PCR product.
  • first-reaction PCR product is a acDNA M molecule
  • a nested oligonucleotide primer would hybridize with an internal site located in the anchor primer or the adapter molecule.
  • novel methods and compositions are provided for the rapid isolation and sequence determination of extended or full-length sequences of novel genes.
  • the present invention is based on the generation of a non- cloned, specially adapted cDNA library (acDNA M ) comprising double stranded DNA molecules that have a first captureable moiety at their 3' end and a known adapter sequence at their 5' end.
  • acDNA M non- cloned, specially adapted cDNA library
  • a modified gene-specific primer having a second captureable moiety is used to generate a gene-specific polynucleotide.
  • Both the cDNA library and the gene-specific primer are uniquely modified such that the cDNA library can be separated from the gene-specific sequence, allowing the differential capture of the gene-specific sequence.
  • the present invention eliminates the time consuming cloning and library screening steps associated with conventional methods of library construction and screening. Additionally, the present invention provides an improvement over current PCR RACE methods by producing a purified gene-specific product in sufficient quantity and purity to allow direct sequencing of the gene-specific PCR product. In view of the above, the present invention provides a novel method for isolating a gene-specific polynucleotide.
  • the method comprises: (a) synthesizing a population of double stranded DNA molecules, wherein each strand has a 5' end and a 3' end, using an anchor primer having a first captureable moiety, wherein the anchor primer is at the 3' end of each double stranded DNA molecule; (b) ligating a double stranded adapter molecule to the 5' end of each double stranded DNA molecule; (c) synthesizing a single stranded gene-specific polynucleotide using a gene-specific primer having a second captureable moiety, wherein the captureable moiety of the gene-specific primer is different from the captureable moiety of the anchor primer; (d) purifying the single stranded gene- specific polynucleotide; and (e) amplifying the purified gene-specific polynucleotide using both a gene-specific primer and a primer that hybridizes to either a sequence located in the anchor primer or a sequence loca
  • the first step of the present invention involves the generation of a specially adapted double stranded cDNA library (acDNA M ).
  • the acDNA M library is a population of double stranded cDNA molecules wherein each cDNA molecule has a captureable anchor primer at the 3' end and a specially adapted DNA molecule at the 5' end.
  • a double stranded cDNA library may be produced from a population of mRNA molecules using methods well-known in the art.
  • the population of mRNA molecules used to generate the cDNA library may be isolated from any tissue or cell population, including cells grown in culture.
  • the mRNA is isolated from tissue or cells actively transcribing a potential gene of interest.
  • Methods of extraction of RNA are well-known in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989), vol. 1, ch.7, "Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells", incorporated herein by this reference. Other isolation and extraction methods are also well-known.
  • RNA isolation is performed in the presence of chaotropic agents such as guanidinium chloride or guanidinium thiocyanate, although other detergents and extraction agents can alternatively be used.
  • the mRNA i.e. poly(A) + RNA
  • the mRNA is typically isolated from the total extracted RNA by chromatography over oligo(dT)- cellulose or other chromatographic media that have the capacity to bind the polyadenylated 3'-end portion of mRNA molecules.
  • the cDNA library is prepared using poly(A) + selected mRNA.
  • the poly(A) + selected mRNA is representative for all the expressed genes in the prepared sample.
  • about 2 ⁇ g of mRNA is used to prepare the cDNA library.
  • total RNA may be used to synthesize cDNA.
  • Double stranded cDNAs are prepared from a population of mRNA molecules using a polythymidylate (polydT) primer and reverse franscriptase to generate the first strand cDNA, thereby creating a mRNA:cDNA hybrid.
  • the polydT primer hybridizes to the polyadenylate (polydA) sequence present on the 3' terminus of each mRNA molecule.
  • the first strand of cDNA is synthesized through the process of reverse transcription whereby the enzyme reverse franscriptase adds deoxyribonucleotides to the 3' terminus of the polydT primer (Varmus, Science, 240, 1427-1435 (1988);
  • the second strand cDNA is generated using an RNAse to break the RNA:cDNA hybrid and a DNA polymerase to synthesize a complementary DNA strand from the template DNA strand.
  • the double stranded acDNA M library of the present invention is generated with a modified polydT primer, called a captureable anchor primer, using the well- known methods of cDNA library construction described above.
  • the captureable anchor primer comprises the single stranded sequence 5'- N(N) a (T) b -3', where N is a nucleotide to which a captureable moiety is attached, (N) a is a polynucleotide where a is an integer of from about 15 to about 50, and (T) b is a polythymidylate where b is an integer of from about 12 to about 25.
  • N and N may be any nucleotide independently selected from the group consisting of adenylate (A), guanidylate (G), cytidylate (C), uridylate (U) and thymidylate (T) nucleotides.
  • N and N may also include derivitized or modified nucleotides.
  • N is the terminal nucleotide located at the 5' end, however, N may be an internal nucleotide as long as the attached captureable moiety does not interfere with anchor primer hybridization with the mRNA.
  • the captureable moiety can be any functional group or molecule that, first, can be joined to a nucleotide N and, second, can be attached to a subsfrate or solid-phase support, such that N remains affixed to the solid-phase support by the attachment of the captureable moiety. While the captureable moiety may form any type of attachment with the substrate or solid-phase support, the attachment must be of sufficient strength to maintain the nucleotide N on the solid-phase support.
  • the captureable moiety is attached to the substrate or solid-phase support by affinity attachment or by covalent bonding.
  • suitable captureable moieties include, for example, avidin, streptavidin, neutravidin, biotin, primary amines, primary carboxylates, thiol alcohols, thiol carboxylates, carbonyls, sugars, lipids, and peptides.
  • the captureable moiety is a biotin molecule.
  • Biotin is a vitamin which allows affinity attachment with the avidin family of proteins.
  • the biotin-avidin attachment is the strongest non-covalent attachment known, having an association constant of 10 15 M "1 .
  • the captureable moiety is a primary amine.
  • a primary amine allows covalent attachment, for example, to an N-oxysuccinimide ester (NOS) by displacing the N-oxysuccinimide group, resulting in the formation of a very specific covalent bond.
  • NOS N-oxysuccinimide ester
  • amines can be attached to several other molecules useful for capture onto solid-phase supports, such as those described in U.S. Patent No. 5,677,276, which is incorporated herein by reference.
  • Other molecules that react with amines are known to the skilled artisan and include, for example, imidoesters and N- hydroxysuccinimidyl esters (NHS-esters). Imidoesters react with primary amines, resulting in an amidine bond (Staros et al., J. Biol. Chem., 256:5890-5893 (1981); Browne et al., Biochem. Biophys. Res. Comm., 67:126-132 (1975)).
  • imidoesters examples include dimethyladipimidate-2-HCl (Pierce #20664), dimethylpimelimidate-HCl (Pierce #20666), dimethylsuberimidate-2HCl (Pierce #20668), dimethyl 3,3'-dithiobisproprionimidate-2HCl (Pierce #20665).
  • NHS-esters react with primary and secondary amines, resulting in a covalent amide bond.
  • Suitable NHS-esters include disuccinimidyl glutarate (Pierce #29592), disuccinimidyl suberate (Pierce #21555), bis(sulfosuccinimidyl)suberate (Pierce #21579), dithiobis(succinimidyl proprionate) (Pierce #22585), dithiobis(sulfosuccinimidyl proprionate) (Pierce #21577), ethylene glycobis(succinimidylsuccinate) (Pierce #21565), ethylene glycobis(sulfosuccinimidylsuccinate) (Pierce #21566), disuccinimidyl tartarate (Pierce #20590), disulfosuccimemidyl tartarate (Pierce #20591), bis[2- (succinimidyloxycarbonyloxy)ethyl]sulfone (Pierce # 21554), bis[2- (sulfo
  • NHS-esters include NHS-ester maleimide componds.
  • Suitable NHS- ester maleimide compounds include, for example, succinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate (Pierce #22320), sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane- 1 -carboxylate (Pierce #22322), m- maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce #22310), m- maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Pierce #22312), succinimidyl 4- (p-maleimidophenyl)-butyrate (Pierce #22315), sulfosuccinimidyl 4-(p- maleimidophenyl)-butyrate (Pierce #22317), bismaleimidohexane (
  • Still other compounds that are reactive with primary amines include NHS-ester haloacetyls, NHS-ester pyridyl disulfides, carbodiimides, arylhalides and arylazides.
  • Suitable NHS-ester haloacetyls include N-succinimidyl(4- iodoacetyl)aminobenzoate (Pierce #22325, 22326) and sulfosuccinimidyl(4- iodoacetyl)aminobenzoate (Pierce #22327, 22328).
  • Suitable NHS-ester pyridyl disulfides include l,4-Di-[3'-2'-pyridyldithio(propionamido)butane] (Pierce #21701), 4-succinimidyloxycarbonyl- ⁇ -(2-pyridyldithio)toluene (Pierce #21558, 21458), sulfosuccinimidyl-6-[ ⁇ -methyl- ⁇ -(2-pyridyldithio)-toluamido]hexane (Pierce #21568, 21569), N-succinimidyl-3-(2-pyridyldithio)-propionate (Pierce #21557, 21657,
  • Suitable carbodiimides are l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (Pierce #22980, 22981) and N,N'- dicyclohexylcarbodiimide (Pierce #20320).
  • a suitable arylhalide is 1 ,5-difluoro-2,4-dinitrobenzene (Pierce #21524).
  • arylazides are reactive with amines, including N-5-azido-2- nitrobenzoyloxysuccinimide (Pierce #21551), N-hydroxysuccinimidyl-4- azidobenzoate (Pierce #21560), N-hydroxysulfosuccinimidyl-4-azidobenzoate (Pierce #21561), N-hydroxysuccinimidyl-4-azidosalicylic acid (Pierce #27715), N- hydroxysulfosuccinimidyl-4-azidosalicylic acid (Pierce #27725), sulfosuccinimidyl- (4-azidosalicylamido)-hexanoate (Pierce #27735), p-nitrophenyl-2-diazo-3,3,3- trifluoropropionate (Pierce #20669), 2-diazo-3,3,3-trifluoropropionylchloride (Pierce #206
  • captureable moieties such as sugar groups and thiol groups
  • Compounds reactive with sugar groups such as phenyl azide-hydrazide, can be useful for capture onto solid-phase supports.
  • Compounds reactive with thiol groups include maleimides, haloacetyls and pyridyl disulfides, such as the NHS-ester maleimide, NHS-ester haloacetyl, and NHS-ester pyridyl disulfide compounds provided above.
  • Still other possible captureable moieties include non-selective photoreactive compounds, such as azidobenzoyl Hydrazide (Pierce #21510, 21509), N-[4-azidosalicylamindo)butyl]-3'(2'-pyridyldithio)propionamide (Pierce 21512), p- Azidophenylglyoxal monohydrate, 4-(p-Azidosalicylamido)butylamine, l-(p- Azidosalicylamido)-4-(iodoacetamido)butane (Pierce #21511), and Bis-[ ⁇ -4- azidosalicylamido)ethyl]disulfide (Pierce #21564).
  • non-selective photoreactive compounds such as azidobenzoyl Hydrazide (Pierce #21510, 21509), N-[4-azidosalicylamindo)butyl]-3
  • the captureable moiety may be attached to an anchor primer using methods well-known in the art (see Hermannson, Bioconjugate Techniques (Academic Press, New York, (1996); Wong, S., Chemistry of Protein Conjugation and Cross-linking (CRC Press, Florida (1991)).
  • reagents for incorporating a primary amine into oligonucleotides are commercially available (Clontech, #5203-1, #5207-2, #5202-1) and described in the art (Connolly, B., Nuc. Acid Res., 15:3131 (1987); Agrawal et al., Nuc. Acid Res., 14:6227 (1986); Smith et al., Nuc.
  • reagents for incorporating other captureable moieties, such as a biotin molecule or a thiol functional group, into oligonucleotides are commercially available (Clontech, #5024-1, #5021-1, #5211-1).
  • biotinylated UTPs which are commercially available with different sized linkers (i.e.
  • Biotin- 11-dUTP Enzo Biochemicals; Biotin-21-UTP, Clontech; Biotin- 16-UTP, Boehringer Mannheim) can be used to produce biotinylated nucleic acids (Ampliscribe T7 High Transcription Kit, Epicentre).
  • an anchor primer having an attached captureable moiety may be purchased commercially such as, for example, a biotinylated anchor primer (Gibco Life Technologies, Grand Island, NY) or an amine-modified anchor primer (Midland Certified Reagents, Midland, TX).
  • the captureable moiety may be attached directly to a nucleotide of the anchor primer.
  • the captureable moiety may be attached indirectly to a nucleotide of the anchor primer via a spacer.
  • the captureable moiety may be attached directly or indirectly to the substrate or solid-phase support.
  • the spacer may encompass any suitable means that can be used to link a nucleotide of the anchor primer to the captureable moiety or any suitable means to link the captureable moiety to the solid-phase support. However, the spacer should not adversely affect the function of the anchor primer or the captureable moiety.
  • the polynucleotide of the anchor primer may comprise any nucleotide sequence consisting of A, G, T, U, and C nucleotides, as well as derivatized or modified nucleotides.
  • the anchor primer polynucleotide comprises a sequence that shares minimal homology with any sequence present in the cDNA library.
  • the low homology design is important to minimize non-specific background generation during the gene-specific PCR amplification step discussed below.
  • a sequence that likely shares minimal homology to a sequence in the cDNA library is a rare restriction enzyme site.
  • rare restriction enzyme sites that occur with a maximum frequency of about one site per 10,000 nucleotides (nt) in the mouse genome include: Not I (GCGGCCGC); Sbfl (CCTGCAGG, average restriction 1/15,000 nt); Spe l (ACTAGT, 1/15,000 nt); Sai l (GTCGAC, 1/20,000 nt); Rsr II (CGGA/TCCG, 1/60,000 nt); and Nhe I (GCTAGC, 1/10,000 nt).
  • the polynucleotide of the anchor sequence comprises a sequence containing at least one rare restriction enzyme site having at least six bases. More preferably, the polynucleotide contains a Not I restriction enzyme site.
  • the polynucleotide of the anchor primer comprises a sequence of at least 7 nucleotides.
  • the anchor primer polynucleotide comprises a sequence of from about 10 to about 40 nucleotides. More preferably, the anchor primer polynucleotide comprises a sequence of from about 20 to about 30 nucleotides. Most preferably, the anchor primer polynucleotide comprises a sequence of about 25 nucleotides.
  • the polythymidylate of the anchor primer comprises a sequence of at least 8 thymidylate nucleotides. Preferably, the polythymidylate comprises a sequence of from about 10 to about 30 thymidylate nucleotides.
  • the polythymidylate comprises a sequence of from about 12 to about 25 thymidylate nucleotides. Most preferably, the polythymidylate comprises a sequence of about 18 thymidylate nucleotides.
  • a particularly preferred captureable anchor primer in accordance with the present invention is a captureable anchor primer comprising the sequence 5*- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3' where N is a biotinylated guanidylate (SEQ ID NO:l).
  • Another equally prefe ⁇ ed captureable anchor primer in accordance with the present invention is a captureable anchor primer comprising the sequence 5*- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3* where N is an aminated guanidylate (SEQ ID NO: 2).
  • an anchor primer comprising and RNA polymerase site is useful.
  • a suitable anchor primer comprising a T3 site is SEQ ID NO: 45or SEQ ID NO: 46
  • a suitable adapter molecule having a T7 site is SEQ ID NO: 13 or SEQ ID NO: 15.
  • An acDNA M library constructed using an anchor primer comprising a T3 site can be amplified using co ⁇ esponding anchor amplification primers, e.g., chosen from the group consisting of SEQ ID NO: 47 or SEQ ID NO: 48.
  • an acDNA M library constructed using an anchor primer comprising a T7 site can be amplified using co ⁇ esponding adapter amplification primers chosen from the group consisting of SEQ ID NO: 17, 18, or 19.
  • the 3' end modified cDNAs of the cDNA M library are synthesized from a mRNA template using a captureable anchor primer under conditions for the preparation of double stranded cDNA that are well-known in the art.
  • the reaction conditions for one prefe ⁇ ed anchor primer is described in Example 1.
  • the captureable anchor primer 5'- N(N) a (T) b -3', hybridizes via its polydT sequence to the polydA sequence present on the 3' terminus of each mRNA molecule and serves as a template for first strand cDNA M synthesis.
  • the first cDNA M strand is synthesized in the 5' or antisense orientation using a reverse franscriptase.
  • Suitable reverse transcriptases include those from avian myeloblastosis virus (AMV) and Moloney murine leukemis virus (MMLV).
  • the second cDNA M strand is generated using an RNAse and DNA polymerase to synthesize the complementary cDNA strand from the first cDNA M strand, resulting in a double-stranded cDNA M having a captureable anchor primer at its 3' end.
  • Suitable RNAses include, for example, RNAse H and RNAase One (Promega, #M4261).
  • Suitable DNA polymerases include E.coli DNA Polymerase I (Promega).
  • a biotinylated anchor primer is used to generate a 3' end biotinylated cDNA library (cDNA B ).
  • an aminated anchor primer is used to generate a 3' end aminated cDNA library (CDNA A ).
  • the captureable anchor primer selected for first sfrand cDNA production is dependent upon the purification procedure chosen to purify the gene-specific sequence (discussed below). It is critical to the present invention that the captureable anchor primer be incapable of capture by the procedure used to capture the gene- specific sequence.
  • the 3' end (mRNA sense) of each double sfranded DNA molecule in the cDNA library will be modified with a captureable moiety resulting in a modified double-stranded cDNA library (cDNA M ). This modification allows the capture of the complete cDNA M library through the attachment of the captureable moiety to a substrate or solid-phase support.
  • a biotinylated cDNA library (cDNA B ) may subsequently be captured through attachment to an avidin-coated substrate. Accordingly, the selection of a biotinylated anchor primer and subsequent generation of a cDNA B library is useful for the purification of a gene-specific sequence having a captureable moiety that does not form an attachment with avidin.
  • An aminated cDNA library (cDNA A ) may subsequently be captured, for example, through attachment to an N- oxysuccinimide ester (NOS) activated substrate. Accordingly, the selection of an aminated anchor primer is useful for the purification of a gene-specific sequence having a captureable moiety that does not form an attachment with NOS.
  • a double stranded adapter molecule is ligated to each cDNA M thereby generating an adapted cDNA M (acDNA M ).
  • the double stranded adapter molecule may comprise any polynucleotide sequence consisting of A, G, T, U, and C nucleotides as well as derivatized and modified nucleotides.
  • the adapter molecule shares minimal homology with the anchor primer sequence.
  • the double stranded DNA adapter molecule preferably comprises a sequence which shares minimal homology with any sequence present in the cDNA library.
  • the low homology adapter sequence design is important to minimize non-specific background generation during the gene-specific PCR amplification step.
  • the adapter molecule may be designed such that it comprises a sequence containing at least one rare restriction enzyme site as described above.
  • the number of rare restriction sites used to construct the adapter sequence should be based on the frequency with which the restriction enzyme site occurs in the genome of the relevant species. For example, a single rare restriction enzyme site sequence may be used to construct the adapter molecule when the restriction enzyme site has eight or more bases.
  • the adapter molecule preferably comprises a sequence containing two or more rare restriction enzyme sites that are sequentially a ⁇ anged and preferably contiguous. More preferably, the adapter molecule comprises a sequence containing about five rare restriction enzyme sites that are sequentially a ⁇ anged and contiguous. Examples of suitable restriction enzymes sites are Not I. Asc l.
  • An example of a prefe ⁇ ed adapter molecule comprises a sequence containing the Sbfl. Spe L Sai l. Rsr II. and Nhe l restriction enzyme sites which are sequentially a ⁇ anged.
  • the double stranded adapter molecule may also be designed such that it comprises the sequence of an RNA polymerase promoter site.
  • the RNA polymerase promoter site is the consensus sequence of the promoter site to which an RNA polymerase binds to initiate RNA synthesis. Suitable RNA polymerase promoter sites include the T7, T3 and SP6 RNA polymerase promoter sites.
  • a preferable RNA polymerase promoter site is the T7 RNA polymerase promoter site.
  • the adapter molecule comprises the sequence of an RNA polymerase promoter site and at least one rare restriction enzyme site.
  • suitable rare restriction enzyme sites include those listed previously.
  • the adapter molecule comprises the sequence of an RNA polymerase promoter site and two or more rare restriction enzyme sites.
  • the adapter molecule may comprise the sequence of theT7 polymerase promoter site and Sbfl. Spe L Sai l. Rsr II and Nhe l restriction enzyme sites.
  • the double sfranded adapter molecule comprises the sequence 5' - Sbfl -T7 promoter sequence - Spe l - Sai l - Rsr l - Nhe l. co ⁇ esponding with the sequence of 5' -
  • the double stranded adapter molecule comprises the sequence of 5' -ACGAGCGGATAACAATTTCACACAGGGCGG- CCGCTAATACGACTCACTATAGGGGTCGAC-3' (SEQ ID NO: 15) and the complementary sequence (SEQ ID NO: 16).
  • the adapter molecule comprises a sequence of at least 15 nucleotides. More preferably, the adapter molecule comprises a sequence of from about 20 nucleotides to about 70 nucleotides. Most preferably, the adapter molecule comprises a sequence of about 60 nucleotides.
  • the double stranded adapter molecule is preferably constructed such that it has a cohesive end at its 5' end and a blunt end at its 3' end as shown in Fig. 2.
  • This construction allows the adapter molecule to be ligated to the cDNA M molecule in the proper orientation and also prevents multiple adapter ligations to one cDNA M molecule.
  • the duplex adapter molecule may be prepared by synthesizing complementary single sfrand adapter polynucleotides such that one of the single-strands is from about four nucleotides to about ten nucleotides longer than its complementary sfrand. The two single strands are subsequently annealed, producing a 5' end overhang.
  • the single strand adapter has a sequence of 5'-ATAGCCTGCAGGTAATACGACTCACTATAGGGA- CTAGTCGACGGACCGCTAGCATCAGATC - 3* (SEQ ID NO: 13) and the reverse complementary single strand has a sequence of 5' -GATCTGATGCTAGCGGTCCG- TCGACTAGTCCCTATAGTGAGTCGTATTACCTGCAGG- 3' (SEQ ID NO: 14) producing a four nucleotide 5' end overhang when the single strands are annealed.
  • the 3' blunt end of the adapter molecule is ligated to the 5' blunt end of each modified double stranded cDNA M molecule using a blunt-end ligase and blunt-end ligation conditions well-known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989).
  • Suitable blunt-end ligases include T4 ligase.
  • the adapter molecule is ligated to the cDNA M molecules using T4 ligase. The captureable moiety at the 3 '-end of the cDNA M molecule prevents ligation of the adapter molecule to the 3' end of the cDNA M .
  • the double stranded adapter molecule may be ligated to modified cDNAs having a captureable anchor primer which is biotinylated (cDNA B ), creating an adapted biotinylated cDNA library (acDNA B ).
  • cDNA B biotinylated cDNA library
  • a double stranded adapter molecule comprising the sequence 5' - ATAGCCTGCAGGTAATACGACTCACTATAGGGACTAGTCGACGGACCGCT AGCATCAGATC - 3' and the complementary sequence (SEQ ID NO: 13 and 14) may be ligated to modified cDNAs having a captureable anchor primer comprising the sequence 5'- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3' where N is a biotinylated guanidylate (SEQ ID NO:l).
  • a double stranded adapter molecule comprising the sequence 5' -ACGAGCGGATAACAATTTCAC- ACAGGGCGGCCGCTA ATACGACTCACTATAGGGGTCGAC-3* and the complementary sequence (SEQ ID NO: 15 and 16) may be ligated to modified cDNAs having a captureable anchor primer comprising the sequence 5'-NAATT- CAACTGGAAGCGGCCGCAGGAA(T) 18 -3' where N is a biotinylated guanidylate (SEG ID NO: 1).
  • the double stranded adapter molecule may be ligated to modified cDNAs having a captureable anchor primer that is aminated, creating an adapted aminated cDNA library (acDNA A ).
  • acDNA A adapted aminated cDNA library
  • a double sfranded adapter molecule comprising the sequence 5' -
  • ATAGCCTGCAGGTAATACGACTCACTATAGGGACTAGTCGACGGACCG- CTAGCATCAGATC - 3' and the complementary sequence (SEQ ID NO: 13 and 14) may be ligated to modified cDNAs having a captureable anchor primer comprising the sequence 5' - NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 - 3' where N is an aminated guanidylate (SEQ ID NO: 2).
  • a double stranded adapter molecule comprising the sequence 5' - ACGAGCGGATAACAATTTCACACAG- GGCGGCCGCTAATACGACTCACTATAGGGGTCGAC - 3' and the complementary sequence (SEQ ID NO: 15 and 16) may be ligated to modified cDNAs having a captureable anchor primer comprising the sequence 5'- NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 - 3* where N is an aminated guanidylate (SEQ ID NO: 2).
  • the complete acDNA M library may be amplified using well-known PCR methods.
  • the library amplification can increase the total amount of the non- cloned acDNA M library.
  • An additional advantage of the total acDNA M amplification is the up-regulation of rare transcripts that subsequently allow easier detection and manipulation of rare cDNA molecules.
  • the acDNA M library may be amplified using a set of two nested oligonucleotide amplification primers having opposing orientation, one of which is located in the adapter molecule sequence and the other of which is located in the anchor primer sequence.
  • the adapter amplification primer (sense) hybridizes with an antisense adapter molecule sequence and is extended in the 3' or sense orientation of the cDNA.
  • the anchor amplification primer hybridizes with a sense anchor primer sequence and is extended in the 5' or antisense orientation of the gene.
  • the adapter amplification primer (Ap) and anchor amplification primer (An) used to amplify the acDNA M library are each preferably an oligonucleotide primer of sufficient length to act as a template for the synthesis of extension products.
  • the Ap or An primer is an oligodeoxynucleotide of at least 12 nucleotides. More preferably, the Ap or An primer is an oligodeoxynucleotide of from about 15 nucleotides to about 50 nucleotides. Most preferably, the Ap or An primer is an oligodeoxynucleotide of from about 20 nucleotides to about 40 nucleotides.
  • the Ap and An amplification primers may be prepared using any suitable method known in the art or may be purchased commercially (MWG Biotech, Inc., Highpoint, N.C.).
  • the oligonucleotide primers may be prepared using the well-known phosphotriester, phosphoramidite, and H-phosphonate methods or automated embodiments thereof, as described in Engels et al., Angew. Chem. Intl. Ed., 28, 716-734 (1989).
  • diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters, 22, 1859-1962 (1981).
  • oligonucleotide primers may be isolated from a biological source, such as a restriction endonuclease digest.
  • the Ap and An amplification primers are selected to be fully or substantially complementary to the different strands of the acDNA M in the regions of the adapter molecule and anchor primer sequences, respectively.
  • Two single stranded nucleotide molecules are substantially complementary when the nucleotides of one strand, optimally aligned with appropriate nucleotide insertions, deletions or substitutions, pair with at least about 80% of the nucleotides of the other strand, preferably, at least about 90% to 95%, and more preferably, from about 98% to about 100%.
  • the Ap and An amplification primers are fully complementary to the respective adapter molecule and anchor primer sequences.
  • any adapter molecule and anchor primer sequence can be selected for use as a primer, care should be taken to avoid sequences that have significant secondary structure, particularly at the 3' end of the primer.
  • primers should be selected which do not have the potential to form primer-dimer structures or be self- complementary.
  • Computer programs, such as Squiggles or Circles are useful for revealing these structures (see also, Rychlik, W., J. NIHRes., 6, 78 (1994).
  • the adapter molecule and anchor primer sequences used for amplification should not be complementary with each other.
  • the complete acDNA M library may be amplified using one Ap amplification primer which is fully or substantially complementary to an antisense sequence located in the adapter molecule and a second An amplification primer which is fully or substantially complementary to a sense sequence located in the anchor primer.
  • the An amplification primer must have at its 5' end the same captureable moiety as the anchor primer. For example, in one prefe ⁇ ed embodiment shown in Fig.
  • one Ap primer preferably comprises 5'- CCTGCAGGTAATACGACTCACTATAGG -3' (SEQ ID NO: 17).
  • a different Ap primer preferably comprises 5'-TAGTCGACG- GACCGCTAGCATCAGATC - 3' (SEQ ID NO: 18).
  • one Ap primer preferably comprises the sequence 5'-ACGAGCG- GATAACAATTTCACACAGGGCGGCCGCTAATACGACTCACTATAGGGGTC GAC-3' (SEQ ID NO: 15).
  • a prefe ⁇ ed amplification primer comprises 5'-NAATTCAACTGGAAGC- GGCCGCAGGAAT -3' (SEQ ID NO: 20), where N is a biotinylated G nucleotide.
  • the anchor primer comprises the sequence 5* -NAATTCAACTGGAAGCGGCCGCAGGAA(T) 18 -3' (SEQ ID NO: 1 or 2)
  • one An primer preferably comprises 5'-NAATTCAACTGGAAGCGGCCGCAGGA -3' (SEQ ID NO: 21), whereN is an aminated G nucleotide.
  • the cDNAs are amplified using PCR amplification methods well-known in the art (see, e.g. Erlich, PCR Technology, Chap. 1, Stockton Press, New York, (1989); Rapley, R. and Walker, L, Molecular Biomethods Handbook, Humana Press, Totowa, New Jersey, (1998)).
  • the standard PCR reaction is typically done in a 50 or 100 ⁇ l volume and usually contains, in addition to the template DNA, 10 mM TrisHCl (pH 8.4), 1.5-3.0 mM MgCl 2 , 0.05-0.5 ⁇ M of each primer, 50-200 ⁇ M of each deoxytriphosphate (dATP, dCTP, dGTP, and dTTP), and 2.5 units of Taq polymerase.
  • the PCR reaction may also include 50 mM KC1, gelatin, glycerol, or DMSO.
  • the PCR reaction is preferably done in a 50 ⁇ l volume and contains 10 mM TrisHCl (pH 8.4), 2.0-3.0 mM MgCl 2 , 0.25 ⁇ M of each primer, 200 ⁇ M of each deoxytriphosphate (dATP, dCTP, dGTP, and dTTP), and 2.5 units of Taq polymerase.
  • the amplification is typically performed automatically using a DNA Thermal Cycler. Suitable thermal Cyclers can be obtained from MJ Research, Inc. (Watertown, MA), PE Biosystems (Foster City, CA), Sfratagene (San Diego, CA) and several other suppliers known in the art.
  • Amplification typically consists of repeated cycles made up of three defined steps, termed denaturation (i.e., strand separation), primer annealing, and primer extension.
  • the template DNA i.e., the acDNA M library
  • the temperature is reduced to 40-60 degrees Celsius for about 30-120 seconds to allow the oligonucleotide primers to anneal with the template DNA.
  • an enzymatic primer reaction is carried out at about 68°-72 degrees Celsius for about 1-5 minutes. The three separate stages are usually repeated between 20 and 40 times.
  • the primer annealing and extension can be done in one step at the same temperature (Kim et al., Nuc. Acids Res., 16:8887-8903 (1988).
  • simultaneously annealing and extending primers at a temperature greater than 55 degrees Celsius may improve the specificity of the reaction by minimizing the extension of primers that are mismatched with the template.
  • the temperature at which the annealing (and extension) is done depends on the length and GC content of the primers.
  • the incubation time for the annealing/extension step may vary according to the length of the target gene being amplified.
  • the PCR reaction conditions and amplification parameters are such that optimal amplification of long cDNA molecules is achieved.
  • the extension step time may be increased to 10-20 minutes and the denaturation step time may be decreased so as to rrnnimize depurination of the template DNA.
  • glycerol or DMSO may be added to the reaction mixture to help lower the melting and strand separation temperature, thus minimizing template depurination.
  • reducing the potassium concentration may improve the efficiency of amplification of longer PCR products.
  • the cycling program may be, for example: 94 degrees Celsius for 2 minutes, followed by 20 cycles of 94 degrees Celsius for 45 seconds (strand separation); 68 degrees Celsius for 5 minutes (primer annealing and extension).
  • strand separation a higher temperature for the primer annealing/extension step provides overall greater stringency which promotes primer specificity.
  • allowing longer extension times promotes the synthesis of longer extended sequences and also allows for greater amplification (i.e., the production of a greater number of DNA molecules).
  • the reactions conditions and/or thermal profile of the reaction may be adapted to reflect the lower stability of the primed template, in accordance with known methods. For example, shorter primers and degenerate primers may require lower annealing temperatures.
  • the complete acDNA M library may be amplified using an RNA polymerase.
  • This amplification method involves producing a synthetic RNA (sRNA) library from the acDNA M library using an RNA polymerase to transcribe sRNA molecules in vitro. Since RNA polymerases produce about 100 molecules per one DNA template molecule, use of an RNA polymerase in this manner results in an amplification of the acDNA M library. This molar up-regulation is advantageous in that less starting material in the form of purified polyA + selected mRNA is required for the detection of gene-specific sequences.
  • RNA amplification facilitates the detection of rare mRNA transcripts.
  • This embodiment also provides the possibility to purify the gene specific DNA strand from the synthetic RNA library. Quality control of the adapter ligation process is facilitated by monitoring the amount of transcript driven from the ligated promoter.
  • RNA promoter consensus sequence and co ⁇ esponding RNA polymerase may be used to produce the sRNA library.
  • suitable RNA promoter sequences and RNA polymerases can be found in Watson et al., Molecular Biology of the Gene, 4 th ed., ch. 13-15, Benjamin Cummings Publishing Co., Menlo Park, CA.
  • the adapter molecule comprises an RNA polymerase promoter site selected from the group consisting of T3, T7 and SP6 RNA polymerase promoter sites.
  • the sRNA library is preferably generated using the co ⁇ esponding RNA polymerase selected from the group consisting of T3, T7 and SP6 RNA polymerases.
  • the T7 promoter sequence and T7 RNA polymerase are used to generate the sRNA library.
  • the adapter molecule comprises a sequence containing 5' - TAATACGACTCACTATAGGGA - 3' (SEQ ID NO 22), which is the consensus sequence for the T7 promoter.
  • the adapter molecule comprises the sequence of 5' - AT- AGCCTGCAGGTAATACGACTCACTATAGGGACTAGTCGACGGACCGCTAG CATCAGATC - 3' (SEQ ID NO: 13).
  • the adapter molecule comprises the sequence 5' -ACGAGCGGATAACAATTTCACA- CAGGGCGGCCGCTA ATACGACTCACTATAGGGGTCGAC-3' (SEQ ID NO: 15).
  • the synthetic RNA is synthesized from the acDNA M template using well- known methods of in vitro transcription (Sambrook et al., pp. 10.5, 10.27-10.37). Given that the adapter molecule containing the RNA promoter sequence is at the 5' end of the acDNA M , the synthetic RNA is transcribed in the sense orientation of the gene. Following synthetic RNA production, the synthetic RNA library is separated from the acDNA M library by digestion of the acDNA M molecules using a deoxyribonuclease, such as DNAse I. The synthetic RNA library can be further purified using well-known methods of RNA column chromatography to remove the small DNA fragments. The resultant purified synthetic RNA library acts as the amplified template for gene-specific extended sequence generation discussed below.
  • the second step of the present invention involves the generation of a gene-specific first strand cDNA sequence using a captureable gene-specific primer and either an acDNA M library or a sRNA library.
  • acDNA M or sRNA molecule in their respective libraries comprises a gene-specific sequence (i.e. the gene of interest), a captureable anchor primer at the 3' end and an adapter molecule at the 5' end.
  • a captureable gene-specific primer is hybridized with a known sequence found in the gene-specific sequence of the acDNA M or sRNA molecule and used to synthesize the partial or full-length gene-specific sequence.
  • the gene-specific cDNA sequence is generated with a captureable gene-specific primer employing well-known methods of primer extension. If a sRNA library is used, the gene-specific cDNA sequence is generated with a captureable gene-specific primer through the process of reverse transcription.
  • a captureable gene-specific primer can be synthesized for a gene of interest after the sequence determination of a short fragment of the gene of interest. Such gene-specific sequence determinations may be deduced, for example, from the amino acid sequence. Additionally, gene-specific sequence determinations may be obtained by screening cDNA libraries to obtain short portions of cDNA clones (i.e. expressed sequence tags or "ESTs") or by using existing EST databases (Venter et al., Science, 252, 1651-1656 (1991); Adams et al., Nature, 355, 632-634 (1992)).
  • ESTs expressed sequence tags
  • the captureable gene-specific primer comprises the single stranded sequence 5'- Y(N) C - 3', where Y is a nucleotide to which a captureable moiety is attached and N c is a oligodeoxynucleotide in which c is an integer of from about 17 to about 40.
  • Y may be any nucleotide selected from the group consisting of A, G, C, U and T nucleotides, including derivatized and modified nucleotides.
  • the gene- specific primer has a captureable moiety attached to a 5'-end nucleotide that allows capture of the extended gene-specific sequence.
  • the captureable moiety is attached to the terminal nucleotide although it may be attached to internal nucleotides as well.
  • the captureable moiety refers to any molecule that can be joined to a nucleotide Y and can also be attached to a substrate or solid support, such that Y is maintained on the solid support by the attachment of the captureable moiety. While the captureable moiety may form any type of attachment with the solid support, the attachment must be of sufficient strength to maintain the nucleotide Y on the solid support.
  • the captureable moiety is attached to the substrate or solid support by affinity attachment or by covalent bonding.
  • Suitable captureable moieties include, for example, avidin, streptavidin, neutravidin, biotin, primary amines, primary carboxylates, sugars, thiol alcohols, thiol carboxylates, lipids, and peptides. Examples of suitable captureable moieties include those compounds listed previously.
  • the captureable moiety is a biotin molecule.
  • An equally preferable captureable moiety is that of a primary amine.
  • the captureable moiety may be attached directly to a nucleotide of the gene-specific primer or, alternatively, may be attached indirectly to such nucleotide via a linker using methods well-known in the art (Hermannson, Bioconjugate Techniques (Academic Press, New York, (1996)).
  • a gene-specific primer having a captureable moiety such as a biotinylated gene-specific primer or an aminated gene- specific primer may be purchased commercially.
  • the captureable moiety must be such that it is different from the captureable moiety of the anchor primer found in the acDNA M .
  • the duality of the captureable moieties of the modified anchor primer and modified gene-specific primer allows subsequent solid phase separations of the acDNA M library from the gene-specific sequence.
  • the gene-specific primer may have, for example, an attached primary amine.
  • the gene-specific primer may have, for example, an attached biotin molecule.
  • the oligodeoxynucleotide (N c ) sequence of the captureable gene-specific primer is selected to be fully or substantially complementary to the known sequence of the gene-specific fragment as that term has been previously defined. While the oligodeoxynucleotide sequence of the captureable gene-specific primer may be any nucleotide sequence, the gene-specific primer should have a random nucleotide distribution and should not contain stretches of polypurines or polypyrimidines.
  • the gene-specific primer sequence should be such that the primer will not anneal with the anchor primer or the adapter molecule primer and will not anneal internally to form hairpin loops or any other secondary structure (Erlich, Ed., PCR Technology, Chap. 1, (Stockton Press, New York (1989)).
  • the captureable gene specific primer is preferably a single stranded oligodeoxynucleotide of sufficient length to act as a template for the synthesis of the gene-specific sequence.
  • the exact length of the gene-specific primer and the quantities used will depend on many factors, including temperature, degree of homology and other reaction conditions. As known in the art, the annealing temperature will vary with the GC content of the particular primer and the composition of the hybridization solution.
  • the length of the gene-specific primer is sufficient to avoid non-specific priming under high annealing temperatures (i.e., temperatures above 60°).
  • the gene-specific primer is an oligodeoxynucleotide of at least 17 nucleotides. More preferably, the gene-specific primer is an oligodeoxynucleotide of from about 20 nucleotides to about 40 nucleotides. Most preferably, the gene-specific primer is an oligodeoxynucleotide of from about 25 nucleotides to about 30 nucleotides.
  • the gene-specific primer may be prepared using any suitable known method for preparing single-stranded oligodeoxynucleotides and may be purchased commercially as well (MWG BioTech, Inc., High Point, N.C.)
  • the oligonucleotide primers may be prepared using the well-known phosphotriester, phosphoramidite, and H-phosphonate methods or automated embodiments thereof, as described in Engels et al, Angew. Chem. Intl. Ed., 28, 716-734 (1989).
  • the gene-specific cDNA of interest may be generated by primer extension of the captureable gene-specific primer following hybridization to the known gene-specific cDNA in the acDNA M library.
  • the captureable gene-specific primer is a biotinylated gene-specific primer or an aminated gene-specific primer.
  • the gene-specific sequence may be extended in either the 3' (sense) or 5' (antisense) orientation of the gene, depending on the gene-specific primer design.
  • a gene-specific cDNA extended in the sense orientation will comprise the gene-specific sequence at its 5' end and the anchor primer sequence at its 3' end.
  • a gene-specific cDNA extended in the antisense orientation will comprise the gene-specific sequence at its 5' end and the adapter molecule sequence at its 3' end.
  • a biotinylated gene-specific primer may be constructed such that it hybridizes to the antisense strand of the known gene-specific sequence of the acDNA M molecule, allowing the gene-specific sequence to be extended in the sense orientation of the gene.
  • the resultant biotinylated gene-specific cDNA comprises the biotinylated gene-specific sequence at its 5' end and the anchor primer sequence at its 3' end.
  • a biotinylated gene-specific primer may be constructed such that it hybridizes to the sense strand of the known gene-specific sequence of the acDNA M molecule, allowing the gene-specific sequence to be extended in the antisense orientation of the gene.
  • the resultant biotinylated gene- specific cDNA comprises the biotinylated gene-specific sequence at its 5' end and the adapter sequence at its 3' end (co ⁇ esponds to having the adapter sequence at the 5' end and gene-specific sequence at the 3' end of the gene).
  • Fig. 4 summarizes diagramatically the amplification primers used.
  • the captureable gene-specific primer may be extended with any suitable thermostable DNA polymerase in the presence of dNTPs using well-known primer extension methodology.
  • the amplification of extended gene-specific sequences may be achieved using well-known PCR methods (Erlich, Ed., PCT Technology (Stockton Press, New York (1989)).
  • PCR methods Erlich, Ed., PCT Technology (Stockton Press, New York (1989)
  • a two step linear PCR amplification method changing the temperature from 68 degrees Celsius to 94 degrees Celsius for multiple cycles (i.e. about 35), may be used to increase the production of gene-specific elongated DNA strand synthesis.
  • the elongation time should be long enough to allow synthesis of the complete gene sequence to its 5' or 3' end. Typical elongation times are from about 0.5 minutes to about 15 minutes.
  • the PCR reaction contains a mixture of acDNA M library molecules, the amplified elongated gene-specific single cDNA strands and the gene-specific primers.
  • the gene-specific sequence of interest may be generated by reverse transcription following hybridization of a captureable gene-specific primer to a known gene-specific sequence in the sRNA library.
  • the captureable gene-specific primer is a biotinylated gene- specific primer or an aminated gene-specific primer. Construction of the sRNA library results in a population of single-stranded sRNA molecules synthesized in the sense orientation. Thus, each sRNA molecule comprises a sense strand sequence containing the 5' end adapter molecule, the gene-specific sequence of interest, and the 3' end anchor primer.
  • a captureable gene- specific primer may be constructed which hybridizes to the known gene-specific sequence of the sRNA molecule.
  • the gene-specific cDNA is reverse transcribed in the antisense orientation, such that the resultant gene-specific cDNA comprises the gene-specific sequence at its 5' end and the adapter sequence at its 3' end (co ⁇ esponds to having the adapter sequence at the 5' end and gene-specific sequence at the 3' end of the sense gene).
  • the gene-specific cDNA is reversed transcribed using reverse franscriptase and well-known methods for first strand cDNA synthesis (Sambrook et al., Molecular Cloning: A Laboratory Manual, vol. 2, "Construction and Analysis of cDNA Libraries").
  • Suitable reverse franscriptase include those from avian myeloblastosis virus (AMV) and Moloney murine leukemis virus (MMLV).
  • a prefe ⁇ ed reverse franscriptase is the MMLV reverse franscriptase.
  • the next step of the present invention involves purifying the gene-specific sequence of interest by separating the gene-specific cDNA from the acDNA M or, in other embodiments, the sRNA library.
  • the separation of the gene-specific cDNA from the acDNA M is achieved by the fact that the gene-specific cDNA and the acDNA M library have different captureable moieties, which moieties allow for the differential capture of the gene-specific sequence.
  • the separation of the gene-specific cDNA from the sRNA library is achieved, in part, through the different biochemical properties of the gene-specific cDNA sequence and the sRNA library which allow the sRNA library to be degraded without affecting the integrity of the gene-specific cDNA.
  • A. Purification of the Gene-Specific cDNA from an acDNA M Library The successful purification of a gene-specific cDNA may be accomplished by constructing any combination of captureable acDNA M libraries and captureable gene- specific cDNAs of interest, as long as the combination allows for the differential capture of the gene-specific cDNAs.
  • the gene-specific purification may be achieved by capturing either the gene-specific cDNA or the acDNA M library such that the unbound cDNA population can be separated from the bound or captured cDNA population.
  • the gene-specific purification may be achieved by capturing the gene-specific cDNA and removing the unbound acDNA M library.
  • the gene-specific purification may be achieved by capturing the acDNA M library and obtaining the unbound gene-specific cDNA.
  • the purification of a gene-specific cDNA may be accomplished through a series of capture steps. For example, the purification may be accomplished by first capturing one cDNA population (i.e. gene-specific cDNA or acDNA M library) on a substrate and subsequently capturing the other cDNA population on a second substrate.
  • the purification can be measured in terms of the quality of sequenceable gene-specific material, defined by: (1) the length of the sequence and (2) the number of readable sequences from an ABI 377 sequence trace. The two step procedure produces substantially better results than the one step method, juudged by quality of sequence.
  • the substrate may be any substrate or solid support that has a rigid or semi- rigid surface suitable for capturing the intended molecule.
  • the substrate has a surface chemistry that forms an attachment between the substrate and captureable moiety.
  • the substrate has a surface that can be coated or derivatized such that the coated or derivatized subsfrate forms an attachment with a captureable moiety.
  • the subsfrate may comprise biological, nonbiological, organic, or inorganic materials, or a combination of these. Examples of suitable materials include agarose, nitrocellulose or other membrane materials, glass, modified silicon, (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof.
  • Suitable substrates may be used and will be readily apparent to the ordinary skilled artisan.
  • Substrates may exist as particles, strands, precipitates, gels, sheets, tubing, spheres, beads, pellets, containers, capillaries, films, plates or slides.
  • the solid support takes the form of a multiwell plate, such as a PCR plate, or a small bead or pellet, or other convenient form.
  • a gene-specific cDNA may be purified by constructing a biotinylated acDNA (acDNA B ) library and an aminated gene-specific primer using previously described methods.
  • the aminated gene-specific primer is hybridized with the acDNA B library and used to generate aminated gene- specific cDNA, as previously described.
  • the biotinylated acDNA B can be separated from the aminated gene-specific cDNA by contacting the acDNA B : aminated gene-specific cDNA mixture with a sfreptavidin-coated substrate.
  • biotinylated acDNA B molecules bind to streptavidin, thereby capturing the acDNA B molecules on the sfreptavidin-coated subsfrate and allowing for the separation of the unbound aminated gene-specific cDNA which remains in the liquid phase.
  • Any subsfrate having a surface chemistry that can be derivatized by streptavidin may be used.
  • Suitable streptavidin-coated substrates include, for example, multi-well microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads, superparamagnetic polymer beads, and paramagnetic porous glass particles.
  • a prefe ⁇ ed streptavidin-coated substrate is a suspension of superparamagnetic avidin- and oligodT- coated polymer beads (Dynal, Inc., Lake Sucess, NY) which allows the removal of the bound acDNA B library from the liquid phase by magnetic forces.
  • a second capture step may be performed using a different capture substrate.
  • the unbound cDNA mixture containing aminated gene-specific cDNA and any residual unbound acDNA B may be subsequently contacted with a NOS-coated substrate.
  • the aminated gene-specific cDNA molecules bind to NOS, thus capturing the gene-specific cDNA on the NOS-coated substrate and further separating the aminated gene-specific cDNA from any unbound acDNA B .
  • Suitable NOS-coated substrates include, for example, multi-well PCR plates (DNA-BIND, Costar). Such PCR plates are especially advantageous because the attached gene- specific single stranded cDNA is stable at 4 degrees Celsius or at -20 degrees Celsius and can be used for repeated gene-specific PCR amplifications (see Example 16).
  • a gene-specific (Gs) cDNA may be purified by constructing an aminated acDNA (acDNA ⁇ library and a biotinylated gene-specific primer using previously described methods.
  • the biotinylated gene-specific primer is hybridized with the acDNA A library and used to generate biotinylated gene-specific cDNA, as previously described. Following linear PCR elongation and amplification of the biotinylated gene-specific cDNA, the acDNA A can be separated from the biotinylated gene-specific cDNA by contacting the acDNA A : biotinylated gene-specific cDNA mixture with a sfreptavidin-coated substrate.
  • biotinylated gene-specific cDNA binds to streptavidin, thus capturing the gene-specific cDNA on the streptavidin-coated substrate and allowing for the separation of the unbound aminated acDNA A library from the captured biotinylated gene-specific cDNA.
  • Suitable sfreptavidin-coated substrates are previously described.
  • a prefe ⁇ ed streptavidin-coated substrate is a suspension of superparamagnetic avidin- and oligodT- coated polymer beads (Dynal, Inc., Lake Sucess, NY) which allows the removal of the bound gene-specific cDNAs from the liquid phase by magnetic forces.
  • Another possible sfreptavidin-coated substrate is a streptavidin-coated multi-well PCR plate. These PCR plates are especially advantageous because the attached gene- specific single stranded cDNA is stable at 4 degrees Celsius or at -20 degrees Celsius and can be used for repeated gene-specific PCR amplifications. Additionally, the above purification may be accomplished in two steps. Prior to the above reaction of the acDNA: biotinylated gene-specific cDNA with a sfrepavidin-coated substrate, the mixture may be contacted with a NOS-coated subsfrate to remove the acDNA A library.
  • the discarded acDNA M library can be re-used to isolate additional gene-specific sequences and may also be re-amplified.
  • the successful purification of a gene-specific cDNA may also be accomplished by constructing a sRNA library that can be used as a template for gene- specific sequence extension by reverse transcriptase.
  • a sRNA is a single stranded RNA comprising a gene-specific sequence of interest, a 5' end adapter sequence, and a 3' end anchor sequence.
  • a captureable gene-specific primer that hybridizes to the known gene-specific sequence in the sRNA template, a captureable gene-specific cDNA is reverse transcribed in the antisense orientation.
  • the captureable gene-specific cDNA is purified by reacting the sRNA: captureable gene-specific cDNA mixture with an RNAse to degrade the sRNA library.
  • RNAse Any suitable RNAse may be used to degrade the sRNA library. Examples of prefe ⁇ ed RNAases include RNAse H and RNAse One.
  • the captureable gene-specific cDNA may be further purified using the methods described above.
  • biotinylated gene-specific cDNA may be further purified by contacting the biotinylated gene-specific cDNA and any residual sRNA with a streptavidin-coated substrate.
  • the biotinylated gene-specific cDNA molecules bind to streptavidin, thus capturing the gene-specific cDNA on the streptavidin-coated substrate and allowing for the separation of the unbound sRNA library from the captured biotinylated gene- specific cDNA.
  • Any suitable streptavidin-coated substrates described above may be used to capture the biotinylated gene-specific cDNA.
  • a prefe ⁇ ed streptavidin-coated subsfrate is a suspension of superparamagnetic avidin- and oligodT- coated polymer beads.
  • Another prefe ⁇ ed streptavidin-coated substrate is a streptavidin-coated multi- well PCR plate which can be used for repeated gene-specific PCR amplifications.
  • aminated gene-specific cDNA may be further purified by contacting the aminated gene-specific cDNA and any residual sRNA with a NOS-coated subsfrate.
  • the aminated gene-specific cDNA binds to NOS, thus capturing the gene-specific cDNA on the NOS-coated subsfrate and further separating the aminated gene-specific cDNA from any residual sRNA.
  • the NOS-coated subsfrate is a NOS-coated multi-well PCR plate which can be used for repeated gene-specific PCR amplifications.
  • the above purification methods produce highly purified, gene-specific single sfranded extended cDNA sequences.
  • the extended cDNA is defined at its 5' end by a known gene-specific primer sequence and defined at its 3' end by either a known anchor sequence or a known adapter sequence, depending on the orientation of the sequence extension.
  • the gene-specific cDNA may be amplified by standard PCR methods using a set of two nested primers having opposing orientation, one of which is located in the known gene-specific sequence and the other of which is located in either an adapter sequence or an anchor primer sequence.
  • a gene-specific sequence may be extended in either the 5' (sense) or 3' (antisense) orientation of the gene, depending on the gene-specific primer design.
  • a gene-specific cDNA extended in the sense orientation will comprise a captureable gene- specific sequence at its 5' end and an anchor primer sequence at its 3' end.
  • a gene- specific cDNA extended in the antisense orientation will comprise a captureable gene- specific sequence at its 5' end and an adapter molecule sequence at its 3' end.
  • the gene-specific cDNA comprises a biotinylated known gene- specific sequence at its 5' end and an anchor primer sequence at its 3' end.
  • the gene-specific cDNA comprises an aminated known gene-specific sequence at its 5' end and an anchor primer sequence at its 3' end.
  • prefe ⁇ ed embodiments include a gene-specific cDNA comprising a biotinylated known gene-specific sequence at its 5' end and an adapter sequence at its 3' end, as well as a gene-specific cDNA comprising an aminated known gene-specific sequence at its 5' end and an adapter sequence at its 3' end.
  • a purified gene-specific cDNA comprising a captureable gene- specific sequence at its 5' end and an anchor primer sequence at its 3' end may be amplified using a set of two nested primers, one of which is located in the known gene-specific sequence (sense primer) and the other of which is located ated in the anchor sequence (anti-sense primer). Amplification is achieved through anchor primer extension in the 5' or antisense orientation and gene-specific primer extension in the 3' or sense orientation of the gene.
  • a purified gene-specific cDNA comprising a captureable gene-specific sequence at its 5' end and an adapter molecule sequence at its 3' end may be amplified using a set of two nested primers, one of which is located in the known gene-specific sequence (anti-sense primer) and the other of which is situated in the adapter sequence (sense primer). Amplification is achieved through adapter primer extension in the 3' or sense orientation and gene- specific primer extension in the 5' or antisense orientation of the gene.
  • the gene-specific amplification primer is selected to be fully or substantially complementary, as that term has been defined, to the sense or antisense strand of the known gene-specific sequence depending on the orientation of the amplification.
  • the Gs amplification primer may comprise the same sequence as the gene-specific primer used to generate the single sfranded gene- specific cDNA (without the attached captureable moiety).
  • the An amplification primer (antisense) is selected to be fully or substantially complementary to the sense strand in the region of the anchor sequence.
  • the An amplification primer used to amplify the purified gene- specific cDNA may comprise the same sequence as the An amplification primer used to amplify the acDNA M library.
  • the Ap amplification primer (sense) is selected to be fully or substantially complementary to the antisense strand in the region of the adapter sequence.
  • the Ap amplification primer used to amplify the purified gene-specific cDNA may have the same sequence as the Ap amplification primer used to amplify the acDNA M library.
  • the known gene-specific (Gs), anchor (An) and adapter (Ap) amplification primers used to amplify the gene-specific cDNA sequences are each preferably a single stranded oligodeoxynucleotide of sufficient length to act as a template for the synthesis of extension products.
  • the length of the Gs, An, or Ap primer should be sufficient to avoid non-specific priming under high annealing temperatures.
  • the Gs, An or Ap primer is an oligodeoxynucleotide of at least 18 nucleotides. More preferably, the Gs, An, or Ap primer is an oligodeoxynucleotide of from about 20 nucleotides to about 40 nucleotides.
  • the Gs, An, or Ap primer is an oligodeoxynucleotide of from about 25 nucleotides to about 30 nucleotides.
  • the Gs, An, and Ap amplification primers may be prepared using any suitable method known in the art, including the methods of oligonucleotide synthesis previously discussed.
  • the purified gene-specific sequence comprises a captureable gene-specific sequence and an anchor primer comprising the sequence 5' - NAATTCAACTGGAAGCGGCCGCAGGAA(T) Ig -3' (SEQ ID NO: 1)
  • an anchor primer comprising the sequence 5' - NAATTCAACTGGAAGCGGCCGCAGGAA(T) Ig -3' (SEQ ID NO: 1)
  • an amplification primer is 5'-NAATTCAACTGGAAGCGGCCGCAG- GA -3* (SEQ ID NO: 20 or 21).
  • the purified gene-specific sequence comprises a captureable gene-specific sequence and an adapter molecule comprising the sequence 5' -ATAGCCTGCAGGTAATACGA- CTCACTATAGGGACTAGTCGACGGACCG CTAGCATCAGATC - 3' (SEQ ID NO: 13)
  • one Ap primer is preferably 5*-CCTGCAGGTAATACGACTCACTATAGG -3' (SEQ ID NO: 17).
  • a different Ap primer is preferably 5'- TAGTCGA- CGGACCGCTAGCATCAGATC - 3' (SEQ ID NO: 18).
  • one Ap primer preferably comprises the sequence 5' - CCGCTAA- TACGACTCACTATAGGGGTCGAC-3' (SEQ ID NO: 19).
  • the gene-specific cDNA sequence may be amplified using standard PCR amplification methods known in the art and described previously. This amplification results in the generation of a gene-specific cDNA fragment population where all gene- specific cDNAs have one homogenous end which sequence is defined by the gene- specific primer and one end of heterogeneous length terminating with either the anchor primer or adapter molecule sequence, depending on the amplification primer choice.
  • the heterogeneity in fragment length of the gene-specific cDNA population is due to the generation of truncated cDNA molecules during the initial cDNA M library construction and also the difference in length of the mRNA polyA tail.
  • the present invention also provides a method for the simultaneous isolation and sequence determination of multiple novel genes.
  • the method involves the utilization of several gene-specific primers, each having a captureable moiety at its 5' end and in a separate reaction chamber.
  • the gene-specific primers are attached to a solid substrate via their captureable moieties.
  • the captured gene-specific primers are each hybridized with a cDNA or RNA (sRNA or mRNA) library and used to generate gene-specific first strand cDNAs.
  • sRNA or mRNA cDNA or RNA
  • the method comprises: (a) synthesizing a population of double stranded DNA molecules having a 5' end and a 3' end; (b) ligating a double stranded adapter molecule to the 5' end of each double stranded DNA molecule; (c) attaching at least one gene-specific oligonucleotide primer having a captureable moiety to a solid substrate, wherein the gene-specific primer is attached via the captureable moiety; (d) synthesizing at least one single sfranded gene-specific polynucleotide using the attached gene-specific primer(s); (e) purifying the single- stranded gene-specific polynucleotide(s); and (f) amplifying the gene-specific polynucleotide(s) using a gene-specific primer and an primer that hybridizes to a sequence located in the adapter molecule or anchor primer, depending on the orientation of the gene specific primers.
  • an adapted cDNA library or an adapted sRNA library is constructed.
  • a cDNA library is generated from mRNA with a polydT primer using methods well known in the art.
  • a double sfranded adapter molecule is ligated to each cDNA, thereby generating an adapted cDNA library.
  • Suitable double stranded adapter molecules have been previously described.
  • the adapter molecule is designed such that it comprises a sequence containing at least one rare restriction enzyme site, as that term has been previously described.
  • the double sfranded adapter molecule comprises the sequence of an RNA polymerase promoter site.
  • RNA polymerase promoter site is the T7 RNA polymerase promoter site.
  • the adapter molecule comprises the sequence of SEQ ID NO: 13.
  • the double-stranded adapter molecule comprises the sequence of SEQ ID NO: 15.
  • the double sfranded adapter molecule is preferably constructed such that it has a cohesive end at its 5' end and a blunt end at its 3' end. The 3' blunt end of the adapter molecule is ligated to the 5' blunt end of the cDNA using a blunt end ligase and well-known blunt end ligation conditions.
  • the adapted cDNA library may be amplified using a pair of amplification primers consisting of an anchor amplification primer and an adapter molecule amplification primer and the previously described PCR amplification methods.
  • a modified cDNA M library i.e. a cDNA library having a captureable anchor sequence, or having an anchor sequence without the captureable moiety, can be used.
  • a sRNA library is constructed as previously described.
  • Captureable gene-specific oligonucleotide primers are synthesized for genes of interest after the sequence determination of a short fragment of the genes of interest. Such gene-specific sequence determinations may be deduced, for example, from the amino acid sequence or may be obtained using existing EST databases.
  • the captureable gene-specific primer comprises the single stranded sequence 5'- Y(N) C -3', where Y is a nucleotide to which a captureable moiety is attached and N c is a oligodeoxynucleotide in which c is an integer of from about 17 to about 40.
  • Each of the gene-specific primers has a captureable moiety attached to the 5'-terminal nucleotide that allows capture of the extended gene-specific sequence to a substrate or solid support.
  • the captureable moiety is affixed to the solid support by affinity attachment or by covalent bonding.
  • Suitable captureable moieties have been previously described above and include, for example, avidin, streptavidin, neutravidin, biotin, primary amines, primary carboxylates, sugars, thiol alcohols, thiol carboxylates, lipids, peptides, and the other compounds previously listed.
  • the captureable moiety is a biotin or primary amine.
  • the gene-specific primer is attached to a substrate or solid support via the captureable moiety. Suitable substrate materials and forms have been previously described.
  • a biotinylated gene-specific primer is affixed to a streptavidin-coated subsfrate, such as streptavidin-coated thin wall PCR tube.
  • an aminated gene-specific primer is attached to a NOS-activated thin wall PCR tube.
  • the gene-specific first strand cDNAs are synthesized with the appropriate enzyme using the cDNA or RNA as a template.
  • the adapted cDNA, synthetic RNA molecules are hybridized with the attached gene-specific primers using well- known methods.
  • the captured gene-specific primers are extended with any suitable thermostable DNA polymerase in the presense of dNTPs using previously described primer extension methods.
  • the resultant gene-specific cDNAs each comprise a known gene-specific primer sequence at the 5' end and an adapter sequence at the 3' end.
  • the gene-specific cDNAs are purified by dissociating the cDNA or RNA library.
  • the dissociation of the library from the gene-specific cDNAs is accomplished by heat denaturation and several washing steps. If a sRNA library is used, an additional RNAse treatment step may be employed to remove the sRNA.
  • the modified cDNA library described above can be removed using the suitable capture subsfrate, e.g. aminated gene-specific sequences were captured on a NOS-coated substrate and avidin beads were used to remove a biotinylated library.
  • the purified gene-specific cDNAs are amplified using standard PCR technology with two nested primers, one of which is located in the gene-specific primer and the other of which is located in the adapter sequence.
  • the adapter molecule comprises the sequence 5' - ATAGCCTGCAGGTAATACGACTCACTATAGGGACTAGTCGACGGACCGCT AGCATCAGATC-3' (SEQ ID NO: 13)
  • the adapter amplification primer (antisense) preferably comprises the sequence 5* - CCTGCAGGTAATACGACTCACTATAGG- 3'(SEQ ID NO: 17).
  • the adapter amplification primer preferably comprises the sequence 5* - TAGTCGACGGACCGCTAGCATC-3' (SEQ ID NO: 18).
  • the adapter amplification primer in yet another prefe ⁇ ed embodiment, in which the adapter molecule comprises the sequence 5' - ACGAGCGGATAACAATTTCACACAGGGCGGCCGCTAATACGACTCAC TATAGGGGTCGAC-3' (SEQ ID NO: 15), the adapter amplification primer preferably comprises the sequence 5' -CCGCTAATACGACTCACTATAGGGGTC- GAC-3' (SEQ ID NO: 19).
  • the gene-specific amplification primer is unique to each gene-specific cDNA and may comprise the same sequence as the gene-specific primer used to generate the first strand cDNA.
  • the double-stranded gene-specific cDNAs are present in the liquid phase after completion of the PCR amplification.
  • the single stranded gene-specific cDNA templates remain attached to the solid support which can be washed and stored at -20 degrees Celsius for further use.
  • the method comprises the additional step of sequencing the isolated gene-specific cDNA product.
  • the purified amplified gene-specific cDNA can be directly sequenced from the end containing the gene-specific sequence using the same gene-specific primer as used for the PCR amplification. Typical annealing and sequencing methods are discussed in the Examples. Additionally, if further sequence information is necessary, the gene-specific cDNA can be sequenced using standard primer walking methodology in conjunction with the present invention (see Examples 15 and 16). For example, typically up to about 600-700 nucleotides of direct sequence can be obtained through one sequencing reaction for short, abundant genes (see Fig.
  • Additional sequence information may be obtained by designing a new gene-specific primer co ⁇ esponding with an internal sequence further downstream or upstream of the gene-specific 5' end.
  • the internal gene-specific primer hybridizes with the downstream or upstream sequence, allowing the determination of additional cDNA sequences. This process of using internal gene-specific sequencing primers to elucidate downstream or upstream sequences can be repeated until the complete sequence of the gene-specific cDNA has been determined (see Fig. 10).
  • the full-length of a gene-specific cDNA may be obtained using primer walking in conjunction with the present invention.
  • the gene-specific cDNA may be truncated at its 5' end rather than embody a full-length sequence.
  • the co ⁇ esponding full-length cDNA may be obtained by repeating the invention using a modified gene-specific primer rather than a modified anchor primer.
  • the modified gene-specific primer has a captureable moiety and co ⁇ esponds with an internal sequence further upstream of the 3' end polydA tail.
  • the first cDNA M strand is synthesized in the previously described manner, resulting in an extended single sfrand cDNA comprising additional 5' end sequence (see Example 16).
  • the same gene-specific template can be used for primer walking. Since no cloning steps are involved, the method of the present invention can be completely automated.
  • the automation of the PCR system provides several advantages. First, automation facilitates the determination of the optimal PCR conditions for the extended sequence generation of a particular gene-specific sequence. DNA fragments derived from cDNA libraries or differential cDNA display systems provide only partial gene sequence information and, in most cases, no additional information about the full-length gene size, gene sequence composition or the abundance of its mRNA transcript is known.
  • the purified gene-specific cDNA can be directly size-fractionated.
  • the method comprises the additional step of size-fractionating the isolated gene-specific cDNA product. Suitable methods of size-fractionation include gel electrophoresis in its various forms, capillary elecfrophoresis and column (size) fractionation.
  • the method comprises the additional step of cloning the isolated gene-specific cDNA product into a vector.
  • the vector is an expression vector.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective.
  • the gene-specific sequence should be operatively linked to an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp, pho A and tac promoters, the S V40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs should further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs should preferably include a translation initiating codon at the beginning and a termination codon (UAA,
  • the expression vector should also preferably include at least one selectable marker for propagation in a host.
  • Suitable markers include dihydrofolate reductase, G418 or neomycin resistence for eukaryotic cell culture and tefracycline, kanamycin, or ampicillin resistance for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Samonella typhimurium cells, fungal cells, such as yeast cells, insect cells, such as Drosophila S2 and Spodoptera Sf9 cells, mammalian cells such as CHO, COS, 293, Bowles melanoma cells and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Vectors prefe ⁇ ed for use in bacteria include, for example, pQ ⁇ 70, pQE60, and pQE-9 (Quiagen, Inc.), pBluescript vectors, Phagescript vectors, pNH16A, pNH18A, pNH46A (Sfratagene Cloning Systems, Inc.), and ⁇ frc99a, ⁇ KK223-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).
  • Prefe ⁇ ed eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Sfratagene) and pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • a plasmid vector is introduced into a host cell in a precipitate or in a complex with a charged lipid.
  • infroduction of the vector construct can be effected by calcium phosphate transfection, DEAE-dexfran mediated transfection, cationic lipid-mediated transfection, elecfroporation, fransduction, infection, or other methods known in the art. Such methods are described in standard laboratory manuals such as Davis et al., Basic Methods in Molecular Biology (1986). If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • a polypeptide can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid, extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatoraphy, and lectin chromatography.
  • HPLC high performance liquid chromatography
  • the methods described above are used for the determination of novel gene sequences without prior knowledge of mRNA length and abundance. Most of the experimental steps are PCR based and therefore directly dependent on the parameters influencing the quality of PCR amplification, such as template concentration, buffer conditions, cycling temperature, cycle number, and cycle duration.
  • the present invention can be also be performed using a multi-well matrix, which would allow standardization of the PCR protocolfor many different genes simultaneously.
  • the present invention provides novel oligonucleotide primer sequences and compositions of oligonucleotide primer sequences which can be used to practice prefe ⁇ ed embodiments of the invention.
  • a novel anchor primer comprising SEQ ID NO: 1 is provided which can be used to generate biotinylated cDNA B using an mRNA template.
  • a novel anchor primer comprising SEQ ID NO: 2 is provided which can be used to generate aminated cDNA A .
  • two novel double-stranded adapter molecules comprising SEQ ID NO: 13 and SEQ ID NO: 15 and their respective complementary sequences comprising SEQ ID NO: 14 and SEQ ID NO: 16, which can be used to generate an adapted acDNA M library.
  • novel amplification primers which can be used to amplify the adapted aCDNA M library are provided.
  • Such primers include anchor amplification primers comprising SEQ ID NO: 20 and SEQ ID NO: 21 and adapter amplification primers comprising SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 and 44.
  • amplification primers which can be used to amplify a gene-specific sequence. These include an anchor amplification primer comprising SEQ ID NO: 23 and above described adapter amplification primers (SEQ ID NO: 17-19).
  • the present invention also includes those oligonucleotide sequences which are substantially identical to the sequences of SEQ ID NO: 1-23, as that term has been defined.
  • the oligonucleotide sequences may also include those sequences capable of hybridizing, under stringent hybridization conditions, to sequences that are complementary to the sequences of SEQ ID NO: 1-23, as those conditions have been defined.
  • the present invention also provides a composition comprising one or more of the novel DNA primer sequences described above and an aqueous carrier.
  • the composition may comprise an anchor primer having a captureable moiety, an adapter molecule, an anchor amplification primer, an adapter amplification primer or any combination thereof.
  • Any suitable aqueous carrier can be used in the composition.
  • the carrier renders the composition stable at a desired temperature, such as room temperature or storage temperature (i.e., 4 degrees Celcius to -20 degrees Celsius), and is of approximately neutral pH. Examples of suitable carriers are known to those of ordinary skill in the art and include Tris-EDTA Buffer, and DEPC-H 2 O.
  • the present invention provides an article of manufacture comprising an anchor primer having captureable moiety and an attached substrate.
  • the anchor primer is a biotinylated anchor primer or an aminated anchor primer.
  • the substrate is an avidin substrate or a NOS-coated substrate.
  • the present invention also includes an isolation and sequencing system, preferably in kit form, for the rapid isolation and sequencing of novel genes from DNA and RNA libraries.
  • the isolation and sequencing system includes, in an amount sufficient to perform at least one assay, a single-stranded anchor primer oligonucleotide having an attached captureable moiety and a double-stranded adapter oligonucleotide as a separately packaged reagent for use in construction of the adapted acDNA M library.
  • the user provides the gene-specific primer oligonucleotides co ⁇ esponding with the gene of interest.
  • the isolation and sequencing system additionally includes a single sfranded amplification anchor primer oligonucleotide and a single stranded amplification adapter primer oligonucleotide as a separately packaged reagent.
  • the user provides the gene-specific primer oligonucleotides co ⁇ esponding with the gene of interest.
  • the PCR primers are utilized in pairs, as is well-known, based on the 5' end sequence of the gene-specific cDNA and the 3' end sequence of the adapter molecule or the 3' end of the anchor sequence.
  • the isolation and sequencing system includes, in an amount sufficient to perform at least one assay, a single stranded anchor primer oligonucleotide having an attached captureable moiety, a double stranded adapter oligonucleotide, a single-stranded amplification anchor primer oligonucleotide, a single sfranded amplification adapter primer oligonucleotide, and at least one single stranded gene-specific primer oligonucleotide having an attached captureable moiety as a separately packaged reagent.
  • the captureable moiety of the anchor primer oligonucleotide is different from that of the gene-specific primer oligonucleotide.
  • At least one captureable gene-specific oligonucleotide having a sequence co ⁇ esponding to that of a known gene or gene fragment, such as an EST is provided.
  • the gene-specific oligonucleotide is desirably obtained from a specific tissue of the user's choice.
  • the gene-specific oligonucleotide may also be obtained from tissue that was treated by exposure to an agent that may alter gene expression, including chemical agents drugs, or growth factors.
  • the isolation and sequencing kit includes at least one gene-specific primer that is captured on a substrate or solid support.
  • Suitable substrates have been previously discussed and include glass, modified silicon, (poly) tetrafluoroethylene, (poly) vinyldendifluoride, polystyrene and polycarbonate materials which exist as particles, strands, precipitates, gels, sheets, tubing, spheres, beads, pellets, containers, capillaries, films, plates, or slides.
  • the gene-specific primers are captured on a PCR plate, a thin-wall PCR tube, or spheres (beads).
  • Prefe ⁇ ed anchor primer oligonucleotides having an attached captureable moiety double stranded adapter oligonucleotides, amplification anchor primer oligonucleotides and amplification adapter primer oligonucleotide are described previously and also described in the Examples.
  • the term "package” refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits an oligonucleotide of the present invention.
  • a package can be a plastic or glass vial used to contain up to microgram quantities of a contemplated oligonucleotide.
  • telomere can be a PCR plate well or other solid phase subsfrate to which microgram quantities of a gene-specific primer oligonucleotide have been operatively affixed, i.e., attached so as to be capable of serving as a template for gene-specific generation and sequencing.
  • Instructions for use typically include a tangible expression describing a sequence of actions to be taken by the user, including at least one method parameter such as the relative amounts of reagent and sample to be admixed, at least one reagent concentration, time periods for which a reagent sample admixture is to be maintained at a specified temperature, or suitable buffer characteristics.
  • the present invention for rapid, high throughput DNA sequencing has several diagnostic and research applications, including genetic and infectious disease diagnosis, toxicology testing, individual identification, genetic research, occupational hazard screening, and agriculture and pharmaceutical development. It is estimated that there are 4000-5000 genetic diseases in humans, in which a mutational change in a gene hinders the function of the gene product, leading to a serious medical condition. Examples of genetic diseases for which assciatedmutations have been identified include cystic fibrosis, phenylketonuria, Alzheimer's disease, cancer, Duchenne muscular dystrophy, and familial hypercholesterolemia. Recent studies show that, while a genetic disease may be associated with one or a few specific mutations, the majority of genetic diseases are likely caused by any of numerous mutations scattered along the affected gene.
  • the present invention can be used to detect any mutation in a target gene, facilitating the diagnosis of genetic diseases as well as the identification of carriers of recessive genetic disorders.
  • the high throughput nature of the present invention and its potential for automation makes it useful as a research tool in population studies aimed at determining which mutations within a target gene are associated with disease and which mutations represent ha ⁇ nless polymorphisms. Such information is instrumental in the development of therapeutic agents.
  • the present invention may be useful in the diagnosis and identification of infectious agents.
  • Each species or strain of micro-organism is expected to have unique DNA sequences which can be used as markers to identify that particular species or strain.
  • rapid high throughput DNA sequencing can determine a particular species or strain of virus or bacteria, facilitating diagnosis and treatment of the infectious disease.
  • the present invention may prove useful in the identification of new species or strains of micro-organisms.
  • DNA typing of individuals for use in forensics and paternity testing. Rapid, high throughput DNA sequencing can be useful for the analysis of polymorphic differences in the DNA of individuals. Furthermore, the potential for automation would allow the simultaneous screening of large numbers of polymorphic markers in the individual. Accordingly, the present invention offers significant advantages over the cu ⁇ ent technique of restriction fragment polymorphism analysis (RFLP), which is time-consuming, labor-intensive and does not provide sequence analysis. DNA typing will also be useful to verify the source and ownership of agricultural products, as new plants and livestock are developed by genetic engineering.
  • RFLP restriction fragment polymorphism analysis
  • the present invention is useful as a research tool for the rapid wide-spread determination of novel full-length DNA sequences and for the characterization of complex multi-gene disorders and other genetic traits.
  • high throughput sequencing can be used in conjunction with known EST sequences and other data generated by the Human Genome Project to determine co ⁇ esponding full-length cDNA sequences.
  • Rapid, high throughput DNA sequencing can also be used to perform repetitive DNA sequence analysis of large numbers of individuals to determine the DNA sequences associated with multi-gene diseases and genetic traits.
  • the present invention can be used for wide-scale sequencing of plant and animal genomes. The sequence data derived from the above sequencing projects will aid in the development of pharmaceutical drugs and the creation of improved crops and livestock.
  • Rapid sequencing can be used to screen the DNA of individuals exposed to high or chronic levels of chemicals or radiation to identify the induced mutations in specific genes as a way of predicting the mutagenic risk of such agents.
  • cDNA M modified cDNA library
  • cDNA M modified cDNA library
  • the resultant cDNA B or cDNA A library is constructed using the ThermoScriptTM System for cDNA Synthesis (Cat. # 11146-016, 11146-032; Gibco Life Technologies, Grand Island, NY).
  • mRNA Isolation is constructed using the ThermoScriptTM System for cDNA Synthesis (Cat. # 11146-016, 11146-032; Gibco Life Technologies, Grand Island, NY).
  • the poly A+ selected mRNA is prepared using standard techniques known in the art. See, e.g., Sambrook, J., et al., Molecular Cloning, A Laboratory Manual , 2d Ed., Cold Spring Harbor Press (1989), pp. 7.1 - 7.29.
  • the isolated poly A+ selected mRNA (about 2 ⁇ g) is reacted with 2 ⁇ l of 500 ng/ ⁇ l 5' biotinylated anchor primer (SEQ ID NO: 1 or 45) (MWG Biotech., Inc.) in a total volume of lO ⁇ l DEPC-H 2 O.
  • the resulting mRNA/ anchor primer mixture is incubated at 70 degrees Celsius for 10 minutes and then quickly chilled on ice to stop the reaction.
  • the mRNA/ anchor primer mixture is centrifuged at 14,000 x g for 10 seconds at 4 degrees Celsius and placed on ice.
  • the first strand reaction mixture is prepared by combining the mRNA/ anchor primer mixture with a mixture comprising the following components that is held on ice and vortexed gently to mix: 4 ⁇ l 5X cDNA Synthesis Buffer (250 mM Tris acetate, pH 8.4), 375 mM potassium acetate, 40 mM magnesium acetate, stabilizer - vortex 5 seconds just prior to use), l ⁇ l 0.1 DTT, l ⁇ l RNaseOUT (40 U/ ⁇ l), 2 ⁇ l lOmM dNTPs, l ⁇ l DEPC H 2 O, l ⁇ l Thermoscript Reverse Transcriptase (15 U/ ⁇ l).
  • the first strand reaction mixture is incubated at 55 degrees Celsius for about 1 hour.
  • the first strand synthesis is terminated by incubating the mixture at 85 degrees Celsius for about 5 minutes.
  • the first sfrand reaction mixture is centrifuged at 14,000 x g for about 10 seconds and placed on ice.
  • the second strand cDNA synthesis is performed by adding the following reaction mixture to the centrifuged first strand reaction mixture: 91 ⁇ l DEPC-H 2 O, 30 ⁇ l 5X Second Strand Buffer (Thermoscript cDNA synthesis kit, BRL), 3 ⁇ l lOmM dNTPs, 1 ⁇ l E. coli DNA ligase (10 units/ ⁇ l), 4 ⁇ l E. coli DNA polymerase I (10 U/ ⁇ l). The resulting mixture is vortexed gently and incubated at 16 degrees Celsius for about 2 hours. Next, 2 ⁇ l (10 units) of T4 DNA Polymerase is added and the reaction mixture is incubated at 16 degrees Celsius for 15 minutes to synthesize the second strand. The second strand synthesis reaction is terminated by the addition of lO ⁇ l of 0.5M EDTA to the reaction mixture, and the result is placed on ice.
  • BRL Second Strand Buffer
  • 3 ⁇ l lOmM dNTPs 3 ⁇ l lOmM dNT
  • the double-stranded cDNA synthesize in the previous step is extracted and purified by adding 150 ⁇ l of Tris-buffered phenol:chloroform:isoamyl alcohol (25:24:1) to the reaction mixture and vortexing for 30 seconds.
  • the reaction mixture is then centrifuged at 16,000 x g for about 5 minutes.
  • the aqueous phase is removed and mixed with 75 ⁇ l 7.5 M NH 4 OAc (2.5 M final) in 550 ⁇ l 100% cold ethanol.
  • the resulting ethanolic mixture is vortexed thoroughly and incubated at about -20 degrees Celsius for at least overnight.
  • the ethanolic mixture is then centrifuged at 16,000 x g at 4 degrees Celsius for sbout 30 minutes.
  • the supernatant is discarded and the cDNA pellet is dried at room temperature until all the residual ethanolic mixture is evaporated.
  • the cDNA is resuspended in the appropriate volume of DEPC-H 2 O to produce a solution of cDNA having a final concentration of 50 ng/ ⁇ l.
  • a cDNA A library is achieved using the same methods of mRNA synthesis, first- and second-strand cDNA synthesis, and cDNA purification, except that an aminated anchor primer (SEQ ID NO: 2, 46) is used for the first-strand cDNA synthesis.
  • an adapted modified cDNA library (acDNA M ), such as an adapted biotinylated or aminated cDNA library (acDNA B or acDNA ⁇ by ligation of a double-stranded adapter molecule to the modified cDNA library.
  • a modified cDNA library such as a biotinylated or aminated cDNA library, is constructed using the methods described in Example 1.
  • the adapter is produced by annealing the oligonucleotides of SEQ ID NO: 13 and 14, creating a double-stranded DNA molecule with one cohesive end (5' sense) and one blunt end (3' sense).
  • the two complementary primers were first denatured at 94 degrees Celsius for 5 minutes, followed by annealing steps through a gradual decrease in temperature from 94 degrees Celsius to 25 degrees Celsius in about 2 hours. The annealing step was performed in a 250mM NaCl solution.
  • the 3' blunt-end of the double-stranded adapter molecule (4.5 ⁇ g) is ligated to the 5' end of the modified double stranded cDNA (1 ⁇ g) using T4 DNA ligase in a total volume of 50 ⁇ l ligase buffer (BRL Biolabs). The reaction is continued for 16 hours at 16 degrees Celsius.
  • An alternative adapter molecule is produced by annealing the oligonucleotides of SEQ ID NO: 15 and 16 using the same annealing conditions. This adapter molecule is blunt-end ligated to a modified cDNA library as described above.
  • EXAMPLE 3 This example demonstrates the amplification of an acDNA M library using anchor amplification primers (An) and adapter amplification primers (Ap) to generate an amplified acDNA M (aacDNA M ).
  • An acDNA M library is made according to the methods described in Examples 1 and 2.
  • the complete acDNA M is amplified using a pair of primers, one of which is located in the anchor sequence and one of which is located in the adapter sequence.
  • an oligonucleotide of either SEQ. ID NO: 17 or 18 is used to amplify an acDNA M having an adapter molecule of SEQ ID NO: 13.
  • an oligonucleotide of either SEQ ID NO: 19 or 44 is used to amplify an acDNA M having an adapter molecule of SEQ ID NO: 15.
  • an oligonucleotide of either SEQ ID NO: 20 or 21 is used to amplify an acDNA M having an anchor primer of SEQ ID NO: l or 2.
  • a small aliquot of the acDNA M library (50 ng) is amplified using the appropriate adapter amplification primer and anchor amplification primer in the following reaction mixture: Platinum Taq Polymerase High Fidelity 1 U (GIBCO BRL cat # 11304-011),1 x High fidelity PCR buffer, 2 mM MgSO4, 0.2 mM dNTP mix.
  • the acDNA M is amplified by PCR at low cycle number and at high annealing temperature using the following 2-step PCR program: 1 cycle: 94°C -2 minutes; 20 cycles: 94°C -45 seconds; 68°C- 5 minutes.
  • the amplified adapted cDNA library (aacDNA M ) is purified using spin columns (Qiagen).
  • the cDNA synthesis, adapter ligation and adapted cDNA amplification can be accomplished within a two-day time period.
  • the cDNA library produced from 2 ⁇ g of polyA+ selected RNA is sufficient to extend the sequence of 100 different DNA fragments.
  • Amplification of the adapted cDNA library produced enough template to extend the sequence of 10,000 different DNA fragments without the need to recreate or amplify the cDNA library.
  • EXAMPLE 4 This example demonstrates the construction of a sRNA library using an acDNA M as a template.
  • An adapted cDNA M library containing either the T3 and /or T7 DNA- dependent RNA polymerse consensus site in either the anchor primer or adapter molecule is used to construct a sRNA library.
  • An acDNA M library is produced according to the methods discussed in Examples 1 and 2; using an anchor primer and/or an adapter molecule having a T3 or T7 site.
  • a suitable anchor primer having a T3 site is SEQ ID NO: 45 or 46.
  • a suitable adapter molecule having a T7 site is SEQ ID NO: 13 or 15.
  • the acDNA M library can be amplified using anchor amplification primers of SEQ ID NO: 47 or 48 and adapter amplification primers of SEQ ID NO: 17, 18, or 19.
  • the acDNA M or aacDNA M library is used as a template for in vitro transcription using the RiboMAX Large Scale RNA production system (Promega, T 7 - #P1300; T 3 - #P1290) according to the manufacturer's instructions.
  • the generated sRNA library is separated from the acDNA M or aacDNA M library template by DNASE I treatment (RQ1 RNAse-free DNAse, Promega #M6101) for one hour at 37°C, followed by a purification step using a CENTRI-SEP spin column (Princeton Separations, #CS-901).
  • EXAMPLE 5 This example demonstrates the generation of an aminated or biotinylated gene- specific first strand cDNA using a synthetic RNA template.
  • the sRNA is made according to the method of Example 4.
  • the sRNA is reacted with 1.5 ⁇ l of lOOng/ ⁇ l aminated or biotinylated gene-specific primer in a total volume of lO ⁇ l DEPC-H 2 O.
  • the annealing reaction is incubated at 70°C for 10 minutes and then quick chilled on ice to stop the reaction.
  • the reaction mixture is then centrifuged at 13,000 x g for 10 seconds at 4°C and placed on ice.
  • the following master reaction mix is prepared on ice and vortexed gently to mix: 4 ⁇ l 5X cDNA Synthesis buffer (250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM magnesium acetate, stabilizer - vortex 5 seconds just prior to use), 1 ⁇ l 0.1 DTT, 1 ⁇ l RNaseOUT (AOxx/ ⁇ ), 2 ⁇ l lOmM dNTPs, l ⁇ l DEPC H 2 O, l ⁇ l Thermoscript Reverse Transcriptase (15 U/ ⁇ l).
  • the master reaction mix (10 ⁇ l) is added to the sRNA on ice.
  • the cDNA synthesis reaction is incubated at 55°C for 1 hour and terminated by incubating the mixture at 85°C for 5 minutes.
  • the first sfrand reaction mixture is centrifuged at 14000 x g for 15 seconds and placed on ice.
  • the gene-specific first strand cDNA is separated from the sRNA library by digesting the RNA using 5 Units of Rnase ONE (Promega cat# M4265) at 37°C for 30 minutes.
  • the gene-specific first strand cDNA is further purified from RNA fragments, enzymes and buffers by spin column purification (Qiagen QIAquick spin column: cat #28104) using the protocol of the manufacturers.
  • the biotinylated or the aminated gene-specific first strand is ready for attachment to a solid phase as described in Example 8 or Example 9.
  • EXAMPLE 6 This example demonstrates the generation of an aminated gene-specific first strand cDNA using an acDNA B or aacDNA B template and an aminated gene-specific primer.
  • Platinum Taq DNA Polymerase High Fidelity was used (Cat. No. 11304-011, Gibco Life Technologies, Grand Island, NY) to generate the animated gene-specific first sfrand cDNA.
  • the single stranded gene-specific cDNA is synthesized in either the sense or antisense orientation depending on the orientation of the gene- specific primer.
  • a gene specific cDNA synthesized from a gene-specific primer having sense orientation contains the anchor sequence.
  • a gene specific cDNA synthesized from a gene-specific primer having antisense orientation contains the adapter sequence.
  • a linear amplification reaction is prepared using 50ng of biotinylated acDNA B or amplified biotinylated aacDNA B library as template, one aminated gene-specific primer (final concentration 200ng/ul) and Platinum Taq Polymerase High Fidelity 1 U, 1 x High fidelity PCR buffer, 2 mM MgSO4, 0.2 mM dNTP mix. (GIBCO BRL cat # 11304-011).
  • the reaction is cycled in a MJ Research Cycler using the following program: 94°C for 2 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 68°C for 5 minutes, (40 cycles); 68°C for 5 minutes; 14°C, forever.
  • the biotinylated adapted cDNA aminated gene-specific first sfrand cDNA is purified from the reaction mix (primers, buffer and enzyme) using QIAquick spin columns (Qiagen: cat #28104).
  • EXAMPLE 7 This example demonstrates the generation of a biotinylated gene-specific first sfrand cDNA using an acDNA A or aacDNA A template and a biotinylated gene-specific primer.
  • Platinum Taq DNA Polymerase High Fidelity was used (Cat. No. 11304-011, Gibco Life Technologies, Grand Island, NY) to generate the biotinylated gene- specific first strand cDNA.
  • a linear amplification reaction is prepared using 50ng of an aminated acDNA A or an amplified aminated aacDNA A library as template, one biotinylated gene-specific primer (final concentration 200ng/ul) and Platinum Taq Polymerase High Fidelity 1 U,l x High fidelity PCR buffer, 2 mM MgSO4, 0.2 mM dNTP mix. (GIBCO BRL cat # 11304-011).
  • the reaction is cycled in a MJ Research Cycler using the following program: 94°C for 2 minutes; 94°C for 30 seconds, 68°C for 5 minutes, (40 cycles); 60°C for 30 seconds, 68°C for 5 minutes; 14°C, forever.
  • the aminated adapted cDNA biotinylated gene-specific first sfrand cDNA is purified from the reaction mix (primers, buffer and enzyme) using QIAquick spin columns (Qiagen: cat #28104).
  • EXAMPLE 8 This example demonstrates the isolation of an aminated gene-specific cDNA from a biotinylated adapted library (acDNA B or aacDNA B ). The aminated gene-specific cDNA is isolated from the reaction products of
  • Example 6 (i.e., a complete acDNA B library and aminated the gene-specific single sfrand cDNA) using two independent purification steps.
  • the first step involves the association of the biotinylated adapted cDNA library to strepatavidin- and oligodT- coated Dynabeads (Dynal) and physical separation of the acDNA B library through magnetic forces from the aminated gene-specific single stranded cDNA.
  • the second step involves the attachment of the aminated gene-specific cDNA to NOS-coated PCR plates and elimination of the remaining acDNA B library molecules through a stringent washing procedure.
  • oligo dT beads (Dynal Dynabeads oligo (dT25) cat# 610.02) are washed and prepared as were used as recommended by the supplier.
  • the aminated gene-specific single sfranded cDNA and acDNA B mixture (product of EXAMPLE 6) is adjusted to 1M NaCl and mixed with lO ⁇ l of the washed Streptavidin/ oligo dT bead suspension.
  • the gene-specific cDNA, acDNA B library, and streptavidin oligo dT bead mixture is incubated at 43°C with moderate shaking for 30 minutes.
  • the beads are centrifuged at 12,000 x g for one minute to pellet the streptavidin/oligo dT beads with the attached aDNA B library.
  • the supernatant containing the aminated gene-specific single sfranded cDNA is removed.
  • the supernatant is purified from the buffer solution using a QIAquick spin column.
  • the aminated gene-specific cDNA obtained in Step 1 is further purified through the solid phase attachment to the wall of a NOS- coated PCR plate well.
  • the aminated gene-specific cDNA is suspended in 50 ⁇ l of 500 mM NaPhosphate, pH 8.5.
  • the gene-specific single stranded cDNA is added to a thin wall PCR plate having a N-oxysuccinimide surface (NOS) (Costar DNA-bind Thermowell M cat# 6573) and incubated at 37 degrees Celsius for 15 minutes (or overnight at 4 degrees Celsius ) .
  • the gene-specific cDNA is blocked at 37 degrees Celsius for 30 minutes using blocking buffer (TTBS, 3% Bovine Serum Albumin, 0.05% Tween, pH 8.0).
  • the blocking agent is removed and the gene-specific cDNA is re-suspended in TE buffer.
  • the plate can be stored at 4 degrees Celsius for future amplification or can be washed for immediate amplification.
  • the attached gene-specific cDNA is washed to eliminate non-bound aDNA B library molecules according to the following wash steps: (1) High wash buffer (3M SSC, 0.1% Sarkosyl) - repeat 3 times; (2) 0.3 NaOH - one time; (3) 0.1 m SSC - one time; (4) High Wash Buffer - repeat twice; and (5) H 2 O - repeat 3 times.
  • EXAMPLE 9 This example demonstrates the isolation of a biotinylated gene-specific cDNA from an aminated adapted library (acDNA A or aacDNA A ).
  • the biotinylated gene-specific cDNA is isolated from the reaction products of Example 7 (i.e., a complete acDNA library and the biotinylated gene-specific single sfrand) by using two independent purification steps.
  • the first step involves the attachment of the aminated acDNA A library to NOS-coated plates and recovery of the biotinylated gene-specific cDNA in solution.
  • the second step involves the association of the biotinylated gene-specific cDNA to sfrepatavidin coated Dynabeads (Dynal) and physical separation from the contaminating aminated acDNA A through magnetic forces.
  • Solid phase attachment of the aminated acDNA A library :
  • the biotinylated gene-specific first sfrand cDNA:acDNA A is suspended in 50 ⁇ l of 500 mM NaPhosphate, pH 8.5. The mixture is added to a thin wall PCR plate having a N-oxysuccinimide surface (NOS) (costar DNA-bind Thermowell M cat# 6573) and incubated at 37 degrees Celsius for about 15 minutes (or overnight at 4 degrees Celsius).
  • NOS N-oxysuccinimide surface
  • Streptavidin Dynabeads (Dynal Dynabeads M-280 Sfrepatividin cat# 112.05/06) are washed and prepared as recommended by the supplier.
  • the product obtained from Step 1 is adjusted to 1M NaCl and mixed with lO ⁇ l of the washed Streptavidin suspension. The mixture is incubated at 43°C with moderate shaking for 30 minutes.
  • the tube containing the mixture of the biotinylated gene-specific cDNA attached to the beads and the aminated acDNA A library are centrifuged at 12,000 x g for one minute.
  • the beads with the attached gene-specific cDNA are resuspended in lOmM TRIS ⁇ H7.5 and can be stored at 4°C.
  • EXAMPLE 10 This example demonstrates the amplification of a purified gene-specific first strand cDNA sequence.
  • a purified gene-specific first strand cDNA is prepared according to the method described in Example 8 or 9.
  • An attached gene specific first strand cDNA directed in the antisense orientation is amplified using an antisense gene-specific primer and a sense adapter sequence primer (i.e., SEQ ID NO: 17, 18, 19, or 44).
  • An attached gene-specific first-strand cDNA directed in the sense orientation is amplified using a sense gene specific primer and an antisense anchor sequence primer (i.e., SEQ ID NO: 23 or 49).
  • the PCR amplification is performed using the Platinum Taq- High Fidelity System (cat # 11304-011).
  • the gene-specific primer 200ng in a 50 ⁇ l reaction and adapter primer (SEQ ID NO: 17, 18, 19, 44) (200 ng) are added to a reaction mixture containing 1 U
  • the reaction is cycled in an MJ Research Cycler using the following program: 94 degrees Celsius for 2 minutes; 94 degrees Celsius 20 for seconds, 55 degrees Celsius for 30 seconds, 68 degrees Celsius for 4 minutes, (35 cycles); 68 degrees Celsius for 5 minutes; 14 degrees Celsius, forever.
  • the supernatant containing soluble amplified gene specific products are purified from dNTPs, enzymes and buffers by spin column purification (Qiagen QIAquick spin column: #28104) using the protocol recommended by the supplier.
  • the gene-specific primer (200ng in a 50 ⁇ l reaction ) and anchor primer (200ng) are added to a reaction mixture containing 1 U Platinum Taq Polymerase High Fidelity, lx High fidelity PCR buffer, 2 mM MgSO 4 , 0.2 mM dNTP mix. (GIBCO BRL # 11304-011).
  • the reaction mixture is added to a PCR well containing an attached aminated gene-specific first sfrand cDNA or to a PCR tube containing a biotinylated gene-specific first-strand cDNA associated with sfrepavidin beads.
  • the PCR cycling program described above is used to amplify the gene-specific cDNA.
  • This example demonstrates the direct sequencing of a PCR product template synthesized from a captured gene-specific first sfrand cDNA.
  • a purified, amplified gene-specific PCR product is generated according to the method described in Example 10.
  • the gene-specific PCR product is cycle sequenced using the PRISMA Sequenase Terminator single-stranded DNA sequencing kit (Applied Biosystem, # 401458).
  • Gene-specific sequencing primers located in the known DNA sequence fragment are used for sequence elongation.
  • the sequencing reaction containing 200-500 ng gene-specific PCR template, 100 ng gene-specific sequencing primer and BigDye terminator are mixed and cycled in a MJ Research Cycler for 31 cycles (96 degrees Celsius for 2 minutes; 96 degrees Celsius for 15 seconds, 50 degrees Celsius for 15 seconds, 60 degrees Celsius for 4 minutes (31 cycles); 14 degrees Celsius, (forever)).
  • EXAMPLE 12 This example demonstrates the rapid sequence determination of a known gene, such as the cyclophilin gene, using the aminated capture approach.
  • a capture modified cDNA B library was constructed using mRNA isolated from whole mouse brain and a biotinylated anchor primer comprising SEQ ID NO: 1.
  • an adapted acDNA B library was constructed by ligating an adapter molecule comprising SEQ ID NO: 13 and 14 to the 5' end of the cDNA B .
  • the acDNA B library was amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 20 and an adapter amplification primer comprising SEQ JO NO: 17.
  • the cyclophilin-specific cDNA was synthesized in the antisense orientation according to the method described in Example 6 using an aminated cyclophilin primer comprising SEQ ID NO: 24.
  • the resultant cyclophilin specific single-stranded cDNA in antisense orientation comprised an aminated cyclophilin-specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the cyclophilin cDNA was purified using the method described in Example 8.
  • the purified cyclophilin cDNA was amplified according to the method described in Example 10 using a cyclophilin-specific amplification primer comprising SEQ ID NO: 50 and an adapter amplification primer comprising SEQ ID NO: 18. Finally, the cyclophilin cDNA was directly sequenced according to the method described in Example 11 using a cyclophilin sequencing primer comprising SEQ ID NO: 29 (see Fig. 8). The sequence obtained by directly sequencing the cyclophilin specific PCR product matched with 100% identity the sequence reported in Genbank (X52803). One sequence read allowed the determination of 509 nucleotides following the cyclophilin specific sequencing primer.
  • EXAMPLE 13 This example demonstrates the rapid sequence determination of the known cyclophilin gene using the biotinylated capture approach.
  • a capture modified cDNA A library was constructed using mRNA isolated from whole mouse brain and an aminated anchor primer comprising SEQ ID NO: 2.
  • an adapted, aminated acDNA A library was constructed by ligating an adapter molecule comprising SEQ ID NO: 15 and 16 to the 5' end of the cDNA A .
  • the acDNA A library was amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 21 and an adapter amplification primer comprising SEQ ID NO: 44.
  • the cyclophilin-specific cDNA was synthesized in the antisense orientation according to the method described in Example 7 using an biotinylated cyclophilin primer comprising SEQ 3D NO: 25.
  • the resultant cyclophilin specific single-stranded cDNA in antisense orientation comprised a biotinylated cyclophilin-specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the cyclophilin cDNA was purified using the method described in Example 9.
  • the purified cyclophilin cDNA was amplified according to the method described in Example 10 using a cyclophilin-specific amplification primer comprising SEQ ID NO: 50 and an adapter amplification primer comprising SEQ ID NO: 44.
  • the cyclophilin cDNA was directly sequenced according to the method described in
  • Example 11 using a cyclophilin sequencing primer comprising SEQ ID NO: 29 (see Fig. 8).
  • the sequence obtained by directly sequencing the cyclophilin specific PCR product matched with 100% identity the sequence reported in GenBank (X52803).
  • One sequence read allowed the determination of 527 nucleotides following the cyclophilin specific sequencing primer.
  • EXAMPLE 14 This example demonstrates the rapid sequence determination of the known cyclophilin gene using the sRNA capture approach.
  • a capture modified cDNA A library was constructed using mRNA isolated from whole mouse brain and an aminated anchor primer comprising SEQ ID NO: 46.
  • an adapted, aminated acDNA A library was constructed by ligating an adapter molecule comprising SEQ ID NO: 13 and 14 to the 5' end of the cDNA A .
  • the sRNA was produced by in vitro transcription with T7 polymerase according to the protocol described in Example 5.
  • the biotinylated cyclophilin-specific cDNA was synthesized in the antisense orientation according to the method described in Example 5 using an biotinylated cyclophilin primer comprising SEQ ID NO: 25.
  • the resultant cyclophilin specific single-stranded cDNA in antisense orientation comprised a biotinylated cyclophilin-specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the cyclophilin cDNA was purified using the method described in Example 9.
  • the purified cyclophilin cDNA was amplified according to the method described in Example 10 using a cyclophilin-specific amplification primer comprising SEQ ID NO: 50 and an adapter amplification primer comprising SEQ ID NO: 44.
  • cyclophilin cDNA was directly sequenced according to the method described in Example 11 using a cyclophilin sequencing primer comprising SEQ ID NO: 29 (see Fig. 8).
  • the sequence obtained by directly sequencing the cyclophilin specific PCR product matched with 100% identity the sequence reported in GenbanK (X52803).
  • One sequence read allowed the determination of 447 nucleotides following the cyclophilin specific sequencing primer.
  • EXAMPLE 15 This example demonstrates the rapid sequence determination of a first unknown gene by extension from a short DNA fragment of known sequence, DST M41, into adjacent sequence regions of unknown sequence, using the aminated capture approach (Fig. 9).
  • a capture modified cDNA B library was constructed using mRNA isolated from whole brain tissue and a biotinylated anchor primer comprising SEQ ID NO: 1.
  • an adapted acDNA B library was constructed by ligating an adapter molecule comprising SEQ ID NO: 13 and 14 to the 5' end of the cDNA B .
  • the acDNA B library was amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 20 and an adapter amplification primer comprising SEQ ID NO: 17.
  • a gene-specific fragment of 268 nucleotides was generated by the TOGA differential display system (U.S.Patent 5,459,037).
  • the fragment contained a polyA stretch at its 3' end, indicating that the DNA fragment was located at the 3' end of a gene. Since no open reading frame could be detected in the DNA fragment it was likely that the cDNA sequence was derived from the 3' untranslated region of a gene.
  • Homology searches to DNA databanks (GeneBank) using the NCBI blast server did not reveal homology to any known gene sequence. In order to learn more about M41, especially concerning the coding region of the gene, extended sequence was generated using the aminated solid phase method described in Example 6, 8,10, 11.
  • An aminated gene-specific primer co ⁇ esponding to the M41 gene-specific fragment was synthesized (SEQ ID NO: 30).
  • the M41 cDNA was generated in the antisense orientation according to the method described in Example 6 using the aminated M41 primer comprising SEQ ID NO: 30.
  • the resultant M41 cDNA comprised an aminated M41 -specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the M41 cDNA was purified using the method described in Example 8.
  • the purified M41 cDNA was amplified according to the method described in Example 10 using a M41- specific amplification primer comprising SEQ ID NO: 32 and an adapter amplification primer comprising SEQ ID NO: 18.
  • the M41 cDNA was directly sequenced according to the method described in Example 11 using an M41 sequencing primer comprising SEQ ID NO: 33.
  • the extended sequence was obtained and could be analyzed within 2 days after starting the experimental protocols.
  • the sequence was extended from the DNA fragment in the antisense orientation by 300 nucleotides of novel sequence, and overlapping the known sequence by 40 nucleotides.
  • Sequence analysis revealed a stop codon that is located 7 nucleotides 5' of the M41 DNA fragment.
  • the extended sequence revealed an open reading frame over the entire extended sequence. This protocol allows rapid sequence extension into the coding region of the gene.
  • the coding sequence can be further explored using DNA and protein analysis tools to determine possible gene functions.
  • EXAMPLE 16 This example demonstrates the rapid sequence extension of a second unknown gene, M51, (Fig. 10) using the aminated capture approach and primer walking on the gene specific PCR product. This example also shows the reusability of the solid phase attached gene specific first sfrand as template for multiple rounds of gene specific PCR amplification.
  • a capture modified cDNA B library was constructed using mRNA isolated from whole brain tissue and a biotinylated anchor primer comprising SEQ ID NO: 1.
  • an adapted acDNA B library was constructed by ligating an adapter molecule comprising SEQ ID NO: 13 to the 5' end of the cDNA B .
  • the acDNA B library was amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 20 and an adapter amplification primer comprising SEQ ID NO: 17.
  • a gene-specific fragment of 295 nucleotides was generated by the TOGA differential display system (U.S.Patent 5,459,037).
  • the sequence was extended towards the 5' end of the gene in the antisense orientation using the aminated solid phase approach described in Example 6, 8, 10 and 11.
  • An aminated M51 primer (SEQ. ID NO: 36) was synthesized, reverse complementary to 26 nucleotides of DST 51. The primer was directed in the antisense orientation and located 135 nucleotides downstream of the 5' end of the DST M51 fragment.
  • the M51 -specific sequence was extended using a biotinylated amplified adapted cDNA library (aacDNA B ) derived from mouse brain poly A+ selected mRNA using the method described in Example 6.
  • aacDNA B biotinylated amplified adapted cDNA library
  • the aminated gene-specific single strand contained 135 nucleotides of the known DST M51 DNA fragment sequence at its 5' end and the adapter sequence (SEQ ID NO: 13) at its 3' end.
  • the M51-gspEXT A was isolated from the contaminating cDNA library (aacDNA B ) using the two consecutive capture protocols described in Example 8, resulting in the covalent attachment of M51-gspEXT A to the wall of a N-oxysuccinimide surface (NOS) PCR plate well (Costar DNA-bind Thermowell M, # 6573).
  • the solid phase attached M51-gspEXT A sequence was amplified using the M51 -specific primer of SEQ ID NO: 37 (antisense orientation) and the adapter amplification primer of SEQ ID. NO: 18 (sense orientation) with the protocol described in Example 10.
  • M51-PCR1 The resultant PCR product, M51-PCR1, was directly sequenced without subcloning using the sequencing primer of SEQ ID NO: 38.
  • the protocols in Examples 6, 8, 10 and 11 were performed in a two day time frame, including the sequence analysis.
  • a new sequencing primer located 120 nucleotides 5' of the DST M51 sequence was synthesized.
  • a new direct sequencing reaction was performed using the M51 -specific PCR product (M51-PCR1) with the sequencing primer of SEQ ID NO: 40.
  • the sequence of M51 was further extended by 406 nucleotides, elongating the DST M51 sequence of 295 by 765 nucleotides within 4 working days.
  • the M51 -specific PCR product M51-PCR1 was used as template for 10 independent direct sequencing reactions using the protocol of Example 11.
  • new M51 gene-specific PCR products can be regenerated by using the solid phase attached gene-specific M51 extended sequence M5 l-gspEXT A as template.
  • the NOS-coated PCR plate containing M51-gspEXT A in one of the 96 wells can be stored in -20 degrees Celsius freezer.
  • PCR plates containing covalently attached gene-specific first strands can be either stored at 4 degrees Celsius in PBS or lyophilized at —20 degrees Celsius for months without damage to the attached DNA.
  • gene-specific DNA has been covalently attached and stored for 13 months without noticeable damage to the DNA.
  • the PCR can be performed using the primers of SEQ ID NO: 37 (anitsense orientation) and SEQ ID NO: 18 (sense orientation) as described above, creating the PCR product M51-PCR1.
  • a new M51 -specific PCR product can be synthesized located further upsfream in the novel extended sequence using the M51-specific primer of SEQ ID NO: 41 (antisense orientation) and the adapter primer of SEQ. ID. 18 (sense orientation).
  • the newly synthesized PCR product, M51-PCR2 is located 78 nucleotides 5' of the DST M51 and contains the known adapter sequence on its 5' end (sense orientation).
  • M51-PCR1 or M51-PCR2 can be used for further extension of the M51 sequence by primer walking.
  • M51 -PCR2 was used as a template for direct sequencing using the primer of SEQ ID NO: 42, located 530 nucleotides upstream (sense) of DST M51.
  • the sequence was further elongated by 405 nucleotides towards the 5' end of the gene, extending the DST M51 by 1170 of novel nucleotides.
  • the sequence was further extended by direct sequencing (Examplel 1), using a sequencing primer of SEQ ID NO: 43, located 695 nucleotides upstream of the DST M51 within the novel sequence generated using sequencing primer SEQ ID NO: 42.
  • the M51 sequence was extended by 1341 nucleotides of novel sequence without the need of subcloning the extended sequences.
  • EXAMPLE 17 This example demonstrates the ability to use multiwell PCR plates in combination with the solid phase direct sequencing protocols for the simultaneous extension of multiple gene fragments. The important parameters can be optimized conveniently in a single 96 well plate.
  • Table 2 below is a diagramatic representation of the use of a 96 well plate to optimize conditions for six templates (DST 1-6), two annealing temperatures in PCR, e.g. 55 degrees Celsius and 60 degrees Celsius, different primers, two different magnesium concentrations (Ml, M2) and different template concentrations.
  • DST 1-6 templates
  • two annealing temperatures in PCR e.g. 55 degrees Celsius and 60 degrees Celsius
  • different primers e.g. 55 degrees Celsius and 60 degrees Celsius
  • Ml, M2 two different magnesium concentrations
  • Each of the multiple gene fragments is defined by its specific sequence composition, its length and its expression profile.
  • DNA fragments derived from cDNA libraries or differential cDNA display systems are only defined by their length (partial gene sequence) and their characteristic sequence. In most cases no additional information on the full length gene size, gene sequence composition or its mRNA abundance is known. This limited information on the gene creates a challenge for the determination of the full-length sequence, especially in a genome that has not been completely sequenced.
  • the variables for the successful sequence extension of a gene is dependent on the abundance of the gene in the adapted cDNA library, its length, the choice of gene specific primers for the generation of the gene specific 1 st sfrand, the primers for the amplification of the gene specific product and the primers for the direct sequencing.
  • the successful amplification of the gene specific sequence by PCR is dependent on the annealing temperature and the magnesium concenfration used for the PCR amplification. All these variables can be easily modified for the optimization of the extended sequence generation simultaneously using a 96 well PCR plate for the experimental design (Table 2). Since no cloning steps are involved and the system can be complete automated and even without prior knowledge of the gene specific parameters optimal sequence extension can be generated for many different genes simultaneously.
  • DST 1 is the cyclophilin gene fragment as described in Example 13
  • DST 2 is the M41 is the gene fragment described in Example 15
  • DST 3 is the M51 is the gene fragment described in Example 16, and DST4, DST 5 and DST 6 are other similar gene fragments.
  • a capture modified cDNA A library is constructed using mRNA isolated from a tissue source such as whole mouse brain and an aminated anchor primer comprising SEQ ID NO: 2.
  • an adapted, aminated acDNA A library is constructed by ligating a double-stranded adapter molecule comprising SEQ ID NO: 13 and 14 to the 5' end of the cDNA A .
  • the acDNA A library is amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 20 and an adapter amplification primer comprising SEQ ID NO: 17.
  • the cyclophilin- specific cDNA is synthesized in the antisense orientation according to the method described in Example 8 using an aminated cyclophilin primer comprising SEQ ID NO: 24.
  • the resultant cyclophilin specific single-stranded cDNA in antisense orientation comprises a aminated cyclophilin-specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the cyclophilin cDNA is purified using the method described in Example 8.
  • the purified cyclophilin cDNA is amplified according to the method described in Example 10 using a cyclophilin-specific amplification primer comprising SEQ ID NO: 50 and an adapter amplification primer comprising SEQ ID NO: 17.
  • the cyclophilin cDNA is directly sequenced according to the method described in Example 11 using a cyclophilin sequencing primer comprising SEQ ID NO: 29.
  • a capture modified cDNA A library is constructed using mRNA isolated from a tissue source such as whole mouse brain and an aminated anchor primer comprising SEQ ID NO: 2.
  • an adapted, aminated acDNA A library is constructed by ligating a double-stranded adapter molecule comprising SEQ ID NO: 13 and 14 to the 5' end ofthe cDNA A
  • a biotinylated gene-specific primer co ⁇ esponding to the M41 gene-specific fragment was synthesized (SEQ ID NO: 30).
  • the M41 cDNA was generated in the antisense orientation according to the method described in Example 6 using the biotinylated M41 primer comprising SEQ ID NO: 30.
  • the resultant M41 cDNA comprised an biotinylated M41 -specific sequence at its 5' end and an adapter sequence at its 3' end.
  • the M41 cDNA was purified using the method described in Example 7.
  • the purified M41 cDNA was amplified according to the method described in Example 10 using a M41-specific amplification primer comprising SEQ ID NO: 32 and an adapter amplification primer comprising SEQ ID NO: 18.
  • the M41 cDNA was directly sequenced according to the method described in Example 11 using an M41 sequencing primer comprising SEQ ID NO : 33.
  • a capture modified cDNA B library was constructed using mRNA isolated from whole brain tissue and a biotinylated anchor primer comprising SEQ ID NO: 1.
  • an adapted acDNA B library was constructed by ligating an adapter molecule comprising SEQ ID NO: 13 to the 5' end of the cDNA B .
  • the acDNA B library was amplified according to the method described in Example 3 using an anchor amplification primer comprising SEQ ID NO: 20 and an adapter amplification primer comprising SEQ ID NO: 17.
  • a gene-specific fragment of 295 nucleotides was generated by the TOGA differential display system (U.S.Patent 5,459,037).
  • the sequence was extended towards the 5' end of the gene in the antisense orientation using the aminated solid phase approach described in Example 6, 8, 10 and 11.
  • An aminated M51 primer (SEQ. ID NO: 36) was synthesized, reverse complementary to 26 nucleotides of DST 51. The primer was directed in the antisense orientation and located 135 nucleotides downstream of the 5' end of the DST M51 fragment.
  • the M51 -specific sequence was extended using a biotinylated amplified adapted cDNA library (aacDNA B ) derived from mouse brain poly A+ selected mRNA using the method described in Example 6.
  • aacDNA B biotinylated amplified adapted cDNA library
  • the aminated gene-specific single sfrand (M51- gspEXT A ) contained 135 nucleotides of the known DST M51 DNA fragment sequence at its 5' end and the adapter sequence (SEQ ID NO: 13) at its 3' end.
  • the M51-gspEXT A was isolated from the contaminating cDNA library (aacDNA B ) using the two consecutive capture protocols described in Example 8, resulting in the covalent attachment of M51-gspEXT A to the wall of a N-oxysuccinimide surface (NOS) PCR plate well (Costar DNA-bind Thermowell M, # 6573).
  • the solid phase attached M51-gspEXT A sequence was amplified using the M51 -specific primer of SEQ ID NO: 37 (antisense orientation) and the adapter amplification primer of SEQ ID. NO: 17 (sense orientation) with the protocol described in Example 10.
  • M51-PCR1 The resultant PCR product, M51-PCR1, was directly sequenced without subcloning using the sequencing primer of SEQ ID NO: 38.
  • the protocols in Examples 6, 8, 10 and 11 were performed in a two day time frame, including the sequence analysis.
  • a new sequencing primer located 120 nucleotides 5' of the DST M51 sequence was synthesized.
  • a new direct sequencing reaction was performed using the M51 -specific PCR product (M51-PCR1) with the sequencing primer of SEQ ID NO: 40.
  • the sequence of M51 was further extended by 406 nucleotides, elongating the DST M51 sequence of 295 by 765 nucleotides within 4 working days.
  • the M51-specific PCR product M51-PCR1 can be used as template for 10 independent direct sequencing reactions using the protocol of Example 11.
  • new M51 gene-specific PCR products can be regenerated by using the solid phase attached gene-specific M51 extended sequence M51 -gspEXT A as template.
  • the NOS-coated PCR plate containing M51-gspEXT A in one of the 96 wells can be stored in —20 degrees Celsius freezer.
  • PCR plates containing covalently attached gene-specific first strands can be either stored at 4 degrees Celsius in PBS or lyophilized at —20 degrees Celsius for months without damage to the attached DNA.
  • gene-specific DNA has been covalently attached and stored for 13 months without noticeable damage to the DNA.
  • the PCR can be performed using the primers of SEQ ID NO: 37 (antisense orientation) and SEQ ID NO: 17 (sense orientation) as described above, creating the PCR product M51-PCR1.
  • a new M51 -specific PCR product can be synthesized located further upstream in the novel extended se ⁇ uence using the M51 -soecific nrimer of SEO ID NO: 41 Canti sense sequence was further extended by direct sequencing (Example 11), using a sequencing pnmer of SEQ ID NO: 43, located 695 nucleotides upstream of the DST M51 within the novel sequence generated using sequencing primer SEQ ID NO: 42.

Abstract

La présente invention concerne des procédés d'isolement et de séquençage rapides de nouvelles séquences génétiques spécifiques. Ladite invention concerne également de nouvelles séquences amorces d'oligonucléotides et leurs compositions ainsi que des kits contenant lesdites séquences amorces d'oligonucléotides et leurs compositions.
PCT/US2001/013807 2000-04-28 2001-04-27 PROCEDES D'ISOLEMENT ET DE SEQUENCAGE RAPIDES DE SEQUENCES GENETIQUES SPECIFIQUES WO2001083696A2 (fr)

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WO2011022970A1 (fr) * 2009-08-28 2011-03-03 Lu Xuedong Amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la pcr et procédé de séquençage utilisant celles-ci
CN102373288A (zh) * 2011-11-30 2012-03-14 盛司潼 一种对目标区域进行测序的方法及试剂盒
US20150211050A1 (en) * 2014-01-27 2015-07-30 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
WO2018053365A1 (fr) * 2016-09-15 2018-03-22 ArcherDX, Inc. Procédés de préparation d'échantillon d'acide nucléique pour l'analyse d'adn acellulaire
US10704082B2 (en) 2016-09-15 2020-07-07 ArcherDX, Inc. Methods of nucleic acid sample preparation
US10718009B2 (en) 2012-05-10 2020-07-21 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
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WO2011022970A1 (fr) * 2009-08-28 2011-03-03 Lu Xuedong Amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la pcr et procédé de séquençage utilisant celles-ci
CN102373288A (zh) * 2011-11-30 2012-03-14 盛司潼 一种对目标区域进行测序的方法及试剂盒
US10718009B2 (en) 2012-05-10 2020-07-21 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US11781179B2 (en) 2012-05-10 2023-10-10 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US20150211050A1 (en) * 2014-01-27 2015-07-30 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US11807897B2 (en) 2014-01-27 2023-11-07 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US10450597B2 (en) 2014-01-27 2019-10-22 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US10683531B2 (en) 2016-09-15 2020-06-16 ArcherDX, Inc. Methods of nucleic acid sample preparation for analysis of cell-free DNA
US10704082B2 (en) 2016-09-15 2020-07-07 ArcherDX, Inc. Methods of nucleic acid sample preparation
US11390905B2 (en) 2016-09-15 2022-07-19 Archerdx, Llc Methods of nucleic acid sample preparation for analysis of DNA
CN110023504B (zh) * 2016-09-15 2023-05-09 阿谢尔德克斯有限责任公司 用于分析无细胞dna的核酸样品制备方法
CN110023504A (zh) * 2016-09-15 2019-07-16 阿谢尔德克斯有限公司 用于分析无细胞dna的核酸样品制备方法
US11795492B2 (en) 2016-09-15 2023-10-24 ArcherDX, LLC. Methods of nucleic acid sample preparation
WO2018053365A1 (fr) * 2016-09-15 2018-03-22 ArcherDX, Inc. Procédés de préparation d'échantillon d'acide nucléique pour l'analyse d'adn acellulaire
US10947582B2 (en) 2016-11-02 2021-03-16 Archerdx, Llc Methods of nucleic acid sample preparation for immune repertoire sequencing

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