Global Amplification Using A Randomly Primed Composite Primer

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.

1 . A method for amplification of a template polynucleotide, comprising:
 (a) incubating a reaction mixture, said reaction mixture comprising:
 (i) a template polynucleotide; 
 (ii) a first primer, wherein the first primer is a composite primer that is hybridizable to a multiplicity of template polynucleotide sites, wherein the composite primer comprises an RNA portion and a 3′ DNA portion; 
 (iii) a DNA-dependent DNA polymerase; and 
 (iv) an RNA-dependent DNA polymerase; 
 wherein the incubation is under conditions that permit first primer random hybridization to the template polynucleotide, and primer extension, whereby a complex comprising a RNA/DNA heteroduplex is generated; and 
   (b) incubating a reaction mixture, said reaction mixture comprising
 (i) at least a portion of the reaction products generated according to step (a); 
 (ii) an amplification primer, wherein said amplification primer is a composite primer comprising an RNA portion and a 3′ DNA portion; 
 (iii) an DNA-dependent DNA polymerase; and 
 (iv) an agent that cleaves RNA from an RNA/DNA hybrid; 
 wherein the incubation is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the primer extension product when its RNA is cleaved and another amplification primer binds to the template and is extended by strand displacement, whereby multiple copies of a polynucleotide amplification product are generated.