Methods of manipulating nucleic acids
First Claim
1. A method of producing a modified nucleic acid probe, comprising:
- contacting a nucleic acid template with a modified random primer under conditions sufficient to permit base-specific hybridization between the template and the primer, wherein the modified random oligonucleotide primer comprises an amine-modified dNTP or a label-substituted dNTP; and
polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template and incorporating at least one modified oligonucleotide primer, thereby producing the modified nucleic acid probe.
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Abstract
Methods are provided for labeling nucleic acid molecules for use in hybridization reactions, and kits employing these methods. The level of labeling is increased by including one or more reactive modifications, such as amine-modifications, into the primers used to initiate synthesis of the nucleic acid molecule, for instance through random-primed reverse transcription. Also provided are modified random primers (such as amine-modified random primers) useful in these methods, labeling and hybridization kits comprising such primers, labeled nucleic acid molecules and mixtures of molecules, and methods for using them. Methods are also provided for amplifying a nucleic acid template contained within extremely small samples, in some cases as little as one cell. In particular embodiments, a single random primer is used for all steps of the amplification method. The nucleic acid template can either be of cellular or viral origin. The disclosure also provides an improved method of fixing cells, tissue sections, or laser microdissected sections from which RNA can be obtained for subsequent use as RNA templates or for generating labeled probe.
38 Citations
46 Claims
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1. A method of producing a modified nucleic acid probe, comprising:
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contacting a nucleic acid template with a modified random primer under conditions sufficient to permit base-specific hybridization between the template and the primer, wherein the modified random oligonucleotide primer comprises an amine-modified dNTP or a label-substituted dNTP; and
polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template and incorporating at least one modified oligonucleotide primer, thereby producing the modified nucleic acid probe. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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- 12. An improved method for random primer reverse transcription labeling of a nucleic acid hybridization probe, the improvement comprising using random primers modified with at least one amine-substituted dNTP or fluorescent-dye modified dNTP in the reverse transcription reaction.
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22. A method of producing an RNA template from a small number of cells, comprising:
lysing a small number of cells by sonication in a buffer, wherein the buffer comprises first strand buffer and an RNase inhibitor, to produce a lysate, wherein the lysate comprises the RNA nucleic acid template. - View Dependent Claims (23, 24)
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37. A method of amplifying a nucleic acid template, comprising:
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contacting a nucleic acid template with a primer under conditions sufficient to permit base-specific hybridization between the template and the primer, wherein the primer comprises a T3-promoter and a random primer and wherein the random primer comprises between about 4 and about 12 nucleotides;
polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template, to produce a polymerized nucleic acid molecule; and
amplifying the polymerized nucleic acid molecule, thereby amplifying a nucleic acid template. - View Dependent Claims (38, 39, 40, 41, 46)
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42. A method of fixing a cell in order to preserve cell structure but permit extraction of high quality RNA for subsequent study, comprising:
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contacting the cell with dithio-bis(Succinimidyl Propionate);
contacting the cell with a reducing agent; and
extracting RNA, thereby fixing a cell to preserve cell structure and permitting extraction of high quality RNA for subsequent study. - View Dependent Claims (43, 44, 45)
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Specification