Global amplification using a randomly primed composite primer
First Claim
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1. A method for amplification of a template DNA, comprising:
- (a) incubating the template DNA with(i) a pool of first composite primers, each first composite primer comprising(1) a 5′
RNA portion that is not hybridizable to the template DNA and(2) a 3′
DNA portion, wherein the 3′
DNA portion of each of the first composite primers comprises a random sequence that is different from the 3′
DNA portion of the other first composite primers in the pool, and(ii) a DNA polymerase having strand displacement activity, whereby a complex comprising an RNA/DNA heteroduplex is generated;
(b) incubating the complex under conditions that permit cleavage of RNA in the RNA/DNA heteroduplex; and
(c) incubating the complex with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′
RNA portion and a 3′
DNA portion, wherein the amplification primer anneals to sequence complementary to the RNA sequence cleaved from the RNA/DNA heteroduplex, whereby multiple copies of an amplification product are generated.
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Abstract
The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.
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Citations
17 Claims
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1. A method for amplification of a template DNA, comprising:
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(a) incubating the template DNA with (i) a pool of first composite primers, each first composite primer comprising (1) a 5′
RNA portion that is not hybridizable to the template DNA and(2) a 3′
DNA portion, wherein the 3′
DNA portion of each of the first composite primers comprises a random sequence that is different from the 3′
DNA portion of the other first composite primers in the pool, and(ii) a DNA polymerase having strand displacement activity, whereby a complex comprising an RNA/DNA heteroduplex is generated; (b) incubating the complex under conditions that permit cleavage of RNA in the RNA/DNA heteroduplex; and (c) incubating the complex with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′
RNA portion and a 3′
DNA portion, wherein the amplification primer anneals to sequence complementary to the RNA sequence cleaved from the RNA/DNA heteroduplex, whereby multiple copies of an amplification product are generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification