NUCLEIC ACID AMPLIFICATION

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.