Global Amplification Using A Randomly Primed Composite Primer

• US 20090068709A1
• Filed: 04/08/2008
• Published: 03/12/2009
• Est. Priority Date: 04/14/2003
• Status: Abandoned Application

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith, Wang, Shenglong
• Application Number:
  US12/099,670
• Publication Number:
  US 20090068709A1
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/203 incorporating a composite nucleic acid containing a polypeptide sequence other than PNA
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

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.

Claims

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.