Nucleic acid amplification

Abstract

The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

What is claimed is: 
     
         1 .- 20 . (canceled) 
     
     
         21 . A method of amplifying RNA sequences, comprising:
 annealing a single-stranded target polynucleotide with a first oligonucleotide that includes a primer region comprising an oligo dT sequence of about 18-21 T residues and a promoter region comprising a T7 promoter sequence to form a first complex;   synthesizing a first strand cDNA by reverse transcription of the first complex to form an RNA/cDNA heteroduplex;   separating the first strand cDNA from the RNA/cDNA heteroduplex;   annealing the first strand cDNA with a plurality of random primers that hybridize at a plurality of positions on the first strand cDNA to form a second complex;   forming a double-stranded cDNA template from the second complex using a combination of DNA dependent polymerase enzymes including exonuclease deficient Klenow and Taq polymerase; and   transcribing the double-stranded cDNA template with an RNA polymerase capable of initiating transcription via said promoter region to produce amplified RNA (aRNA) containing a sequence complementary to the single-stranded target polynucleotide.   
     
     
         22 . The method of  claim 21 , wherein the first oligonucleotide further comprises an anchor sequence between the primer region and the promoter region. 
     
     
         23 . The method of  claim 21 , further comprising degrading the first oligonucleotide remaining after synthesizing a first strand cDNA with an exonuclease. 
     
     
         24 . The method of  claim 23 , wherein the exonuclease is exonuclease I. 
     
     
         25 . The method of  claim 21 , wherein separating the first strand cDNA from the RNA/cDNA heteroduplex comprises denaturing the RNA/cDNA heteroduplex. 
     
     
         26 . The method of  claim 25 , wherein denaturing the RNA/cDNA heteroduplex is done by heating the RNA/cDNA heteroduplex at 95° C. for at least 5 minutes. 
     
     
         27 . The method of  claim 25 , wherein denaturing the RNA/cDNA heteroduplex is done by treating the RNA/cDNA heteroduplex with alkali. 
     
     
         28 . The method of  claim 21 , wherein separating the first strand cDNA from the RNA/cDNA heteroduplex comprises enzymatic degradation of the RNA. 
     
     
         29 . The method of  claim 28 , wherein the enzymatic degradation of the RNA is done using RNase H. 
     
     
         30 . The method of  claim 21 , wherein the random primers comprise at least about six random nucleotides. 
     
     
         31 . The method of  claim 21 , wherein the random primers comprise at least about nine random nucleotides. 
     
     
         32 . The method of  claim 21 , wherein forming the double-stranded cDNA template further comprises:
 adding the combination of DNA dependent polymerase enzymes including exonuclease deficient Klenow and Taq polymerase to the second complex to form an extension reaction mixture;   incubating the extension reaction mixture at room temperature for between about 5 minutes to about 10 minutes;   incubating the extension reaction mixture at a first elevated temperature of about 37° C. for between about 10 minutes to about 30 minutes; and   incubating the extension reaction mixture at a second elevated temperature of about 72° C. for between about 5 minutes to about 15 minutes.   
     
     
         33 . The method of  claim 25 , further comprising after incubating the extension reaction mixture at the second elevated temperature, cooling the extension reaction mixture to 4° C. until transcribing the double-stranded cDNA template to produce amplified RNA is initiated. 
     
     
         34 . The method of  claim 21 , after forming a double-stranded cDNA template, further comprising purifying the double-stranded cDNA template. 
     
     
         35 . The method of  claim 21 , further comprising:
 annealing the aRNA with a third oligonucleotide comprising a primer region operably linked to a promoter region to form a third complex;   synthesizing a first strand of an additional DNA template by reverse transcription of said third complex to produce an aRNA/DNA heteroduplex;   separating the first strand cDNA from the aRNA/cDNA heteroduplex;   annealing the first strand of additional DNA template with the first oligonucleotide to form a fourth complex;   forming additional double-stranded DNA templates from the fourth complex using a combination of DNA dependent polymerase enzymes including exonuclease deficient Klenow and Taq polymerase; and   transcribing the additional double-stranded DNA templates with an RNA polymerase capable of initiating transcription via the promoter region of the first oligonucleotide to produce aRNA that contains sequences complementary to the single-stranded target polynucleotide or via the promoter region of the third oligonucleotide to produce aRNA that contains sequences present in the single-stranded target polynucleotide.