Methods of RNA amplification in the presence of DNA
First Claim
1. A method of selectively generating multiple copies of a polynucleotide sequence of or complementary to target RNA which is in a sample comprising DNA, said method comprising the steps of:
- (a) hybridizing to the target RNA in said sample a first primer comprising a sequence (A) that is not complementary to the target RNA, and a sequence (B) at the 3′
-end which hybridizes to the target RNA;
(b) extending the first primer with at least one enzyme comprising RNA-dependent DNA polymerase activity in the presence of at least one compound comprising DNA-dependent DNA polymerase inhibitor activity, whereby DNA-dependent DNA polymerase activity of the enzyme comprising RNA-dependent DNA polymerase activity is inhibited by said compound, whereby a complex comprising a primer extension product and the target RNA is produced, whereby the first primer extension product comprises a sequence (Y) that is complementary to the target RNA and comprises sequence (A);
(c) disabling or removing at least one compound comprising DNA-dependent DNA polymerase inhibitor activity; and
(d) producing multiple copies of a polynucleotide sequence complementary to the target RNA and/or complementary to sequence (Y) using sequence (A).
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Abstract
The invention provides methods for amplification of RNA. The methods are particularly suitable for specifically amplifying RNA in the presence of DNA. The methods involve producing a marked first primer extension product from a target RNA in the presence of a DNA-dependent DNA polymerase inhibitor, which prevents replication of DNA by the reverse transcriptase enzyme. The marked nucleic acid products are subsequently selectively amplified in the presence on non-marked nucleic acids. The methods are useful for production and analysis of polynucleotide sequences complementary to an RNA sequence. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.
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Citations
46 Claims
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1. A method of selectively generating multiple copies of a polynucleotide sequence of or complementary to target RNA which is in a sample comprising DNA, said method comprising the steps of:
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(a) hybridizing to the target RNA in said sample a first primer comprising a sequence (A) that is not complementary to the target RNA, and a sequence (B) at the 3′
-end which hybridizes to the target RNA;(b) extending the first primer with at least one enzyme comprising RNA-dependent DNA polymerase activity in the presence of at least one compound comprising DNA-dependent DNA polymerase inhibitor activity, whereby DNA-dependent DNA polymerase activity of the enzyme comprising RNA-dependent DNA polymerase activity is inhibited by said compound, whereby a complex comprising a primer extension product and the target RNA is produced, whereby the first primer extension product comprises a sequence (Y) that is complementary to the target RNA and comprises sequence (A); (c) disabling or removing at least one compound comprising DNA-dependent DNA polymerase inhibitor activity; and (d) producing multiple copies of a polynucleotide sequence complementary to the target RNA and/or complementary to sequence (Y) using sequence (A). - View Dependent Claims (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 36, 37, 38, 39)
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2. A method of selectively generating multiple copies of a polynucleotide sequence complementary to a target RNA which is in a sample comprising DNA, said method comprising the steps of:
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(a) hybridizing to the target RNA a first primer comprising a sequence (A) that does not hybridize to the target RNA, and a sequence (B) at the 3′
-end, which hybridizes to the target RNA;(b) extending the first primer with at least one enzyme comprising RNA-dependent DNA polymerase activity in the presence of at least one compound comprising DNA-dependent DNA polymerase inhibitor activity, whereby a complex comprising a first primer extension product and the target RNA is produced, whereby the first primer extension product comprises a sequence (Y) that is complementary to the target RNA and comprises sequence (A); (c) disabling or removing at least one compound comprising DNA-dependent DNA polymerase inhibitor activity; (d) cleaving the target RNA in the complex of step (b); (e) extending a second primer along the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity, wherein the second primer comprises sequence (C) that is complementary to a sequence (C′
) on the first primer extension product to produce a complex comprising the first primer extension product and a second primer extension product, whereby the second primer extension product comprises the sequence (C), a sequence (Y′
) complementary to sequence (Y) and sequence (A′
) complementary to sequence (A);(f) producing multiple copies of a polynucleotide sequence complementary to the target RNA using a primer with a sequence (A) and/or sequence (A′
). - View Dependent Claims (15, 29, 32)
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4. The method of claims l or 2, wherein the method further comprises clonally producing on a solid substrate or in an emulsion multiple copies of a polynucleotide sequence complementary to the target RNA using a primer with a sequence (A) and/or sequence (A′
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40. A method for performing expression analysis of one or more target RNA sequences comprising:
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(a) collecting a sample of nucleic acid comprising RNA and DNA; (b) optionally enriching the nucleic acid in the sample; (c) contacting the nucleic acid with an inhibitor of DNA-dependent DNA polymerase activity; (d) hybridizing to the target RNA a first primer comprising a sequence (A) that is not complementary to the target RNA, and a sequence (B) at the 3′
-end which hybridizes to the target RNA;(e) extending the first primer with at least one enzyme comprising RNA-dependent DNA polymerase activity in the presence of at least one compound comprising DNA-dependent DNA polymerase inhibitor activity, whereby DNA-dependent DNA polymerase activity of the enzyme comprising RNA-dependent DNA polymerase activity is inhibited by the compound, whereby a complex comprising a primer extension product and the target RNA is produced, whereby the first primer extension product comprises a sequence (Y) that is complementary to the target RNA and comprises sequence (A); (f) disabling or removing at least one compound comprising DNA-dependent DNA polymerase inhibitor activity; (g) producing multiple copies of a polynucleotide sequence complementary to the target RNA and/or complementary to sequence (Y) using sequence (A); and (h) further analyzing the amplified products of step (g). - View Dependent Claims (41, 42, 43)
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44. A method of generating at least one double stranded DNA product corresponding to a sequence of a target RNA or a portion thereof comprising:
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(a) adding to a reaction mixture comprising target RNA and further comprising DNA; (i) at least one first primer comprising a 3′
annealing sequence and a 5′
tail sequence;(ii) at least one DNA-dependent DNA polymerase inhibitor; and (iii) an RNA-dependent DNA polymerase having DNA-dependent DNA polymerase activity that is inhibited by the at least one compound that is a DNA-dependent DNA polymerase inhibitor; (b) disabling or removing the at least one DNA-dependent DNA polymerase inhibitor from the reaction mixture; (c) annealing and extending at least one all-DNA second primer comprising an annealing sequence with a DNA-dependent DNA polymerase, wherein said double stranded product is labeled at at least one end; and (d) clonally amplifying one or both strands of the double-stranded product. - View Dependent Claims (45, 46)
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Specification