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 with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′
RNA portion and a 3′
DNA portion, whereby multiple copies of an amplification product are generated, wherein the incubation of the complex with the amplification primer is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the primer extension product when the RNA of the amplification primer is cleaved and another amplification primer binds to the complex and is extended by strand displacement, whereby multiple copies of a polynucleotide 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
27 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 with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′
RNA portion and a 3′
DNA portion, whereby multiple copies of an amplification product are generated, wherein the incubation of the complex with the amplification primer is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the primer extension product when the RNA of the amplification primer is cleaved and another amplification primer binds to the complex and is extended by strand displacement, whereby multiple copies of a polynucleotide amplification product are generated. - 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)
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Specification