Global amplification using a randomly primed composite primer

摘要

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.

主权项

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.