Methods for production and purification of nucleic acid molecules
First Claim
1. A method for making a nucleic acid molecule comprising (a) mixing a nucleic acid template with (i) one or more polypeptides having polymerase activity and/or reverse transcriptase activity and (ii) a primer-adapter nucleic acid molecule;
- and (b) incubating said mixture under conditions sufficient to make a first nucleic acid molecule complementary to all or a portion of said template, wherein said primer-adapter nucleic acid molecule comprises one or more ligands and one or more cleavage sites.
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Abstract
The present invention is directed to methods for the production and isolation of nucleic acid molecules. In particular, the invention concerns isolation of mRNA molecules and the production and isolation of nucleic acid molecules (e.g., cDNA molecules or libraries), which may be single- or double-stranded. Additionally, the invention concerns selection and isolation of particular nucleic acid molecules of interest from a sample which may contain a population of molecules. Specifically the invention concerns affinity-labeled primer-adapter molecules which allow improved isolation and production of such nucleic acid molecules, increasing both product recovery and speed of isolation.
22 Citations
53 Claims
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1. A method for making a nucleic acid molecule comprising
(a) mixing a nucleic acid template with (i) one or more polypeptides having polymerase activity and/or reverse transcriptase activity and (ii) a primer-adapter nucleic acid molecule; - and
(b) incubating said mixture under conditions sufficient to make a first nucleic acid molecule complementary to all or a portion of said template, wherein said primer-adapter nucleic acid molecule comprises one or more ligands and one or more cleavage sites. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 40)
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- 33. A nucleic acid molecule comprising one or more primer-adapter molecules, wherein said primer-adapter molecule comprises one or more ligands and one or more cleavage sites.
- 41. A kit for the production of a nucleic acid molecule comprising one or more containers, wherein a first container comprises a primer-adapter molecule comprising one or more ligands and one or more cleavage sites.
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44. A method for producing a cDNA molecule, said method comprising
(a) mixing an mRNA template with a polypeptide having reverse transcriptase activity and a primer-adapter nucleic acid molecule, said primer-adapter molecule comprising one or more ligands and one or more cleavage sites; -
(b) incubating said mixture under conditions sufficient to make a first DNA molecule complementary to all or a portion of said template, thereby forming a DNA-primer-adapter molecule;
(c) binding said DNA-primer-adapter molecule to a solid support through a ligand-hapten interaction; and
(d) isolating said first DNA molecule from said solid support by cleaving said one or more cleavage sites. - View Dependent Claims (45, 46, 47, 48)
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49. A method for producing a cDNA molecule, said method comprising
(a) incubating an mRNA template with one or more polypeptides having reverse transcriptase activity and with a primer under conditions sufficient to make a first DNA molecule complementary to all or a portion of said template; -
(b) incubating said first DNA molecule with a primer-adapter molecule, wherein said primer-adapter molecule comprises one or more ligands and one or more cleavage sites, under conditions sufficient to form a double-stranded DNA molecule comprising a primer-adapter molecule;
(c) binding said double-stranded DNA molecule to a solid support through a ligand-hapten interaction; and
(d) isolating said double-stranded DNA molecule from said solid support by cleaving said one or more cleavage sites. - View Dependent Claims (50)
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51. A method for isolating a mRNA molecule, said method comprising
(a) mixing a RNA sample with a primer-adapter molecule which hybridizes to mRNA, wherein said primer-adapter molecule comprises one or more ligands and one or more cleavage sites, thereby forming a mRNA-primer-adapter molecule; -
(b) binding said mRNA-primer-adapter molecule to a solid support through a ligand-hapten interaction; and
(c) isolating said mRNA molecule from said solid support by cleaving said one or more cleavage sites. - View Dependent Claims (52)
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53. A method for isolating one or more desired nucleic acid molecules from a population of nucleic acid molecules comprising
(a) mixing said population of nucleic acid molecules with one or more target-specific primer-adapter molecules; -
(b) incubating said mixture under conditions sufficient to bind said primer-adapter molecules to said desired nucleic acid molecules; and
(c) isolating one or more of said desired nucleic acid molecules.
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Specification