Nucleic acid amplification
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.
1 . A method of synthesizing a double stranded cDNA of an RNA molecule, said method comprising
a) annealing an RNA molecule with a first oligonucleotide comprising a primer operably linked to a promoter region to form a first complex,
b) synthesizing a first strand cDNA by reverse transcription of said first complex to form hybrid(s) of RNA and first strand cDNA,
c) degrading first oligonucleotides not used in a) or b) above with exonuclease activity, and
d) annealing said first strand cDNA, after denaturing the hybrid(s) or degrading the RNA from said hybrid(s), with a plurality of second oligonucleotides comprising a random primer region to form a population of second complexes, and
e) forming double stranded cDNA from said population of second complexes with DNA polymerase activity.
2 . The method of claim 1 wherein said RNA is mRNA.
3 . The method of claim 1 wherein said RNA is part of a cellular mRNA preparation.
4 . The method of claim 2 wherein said first oligonucleotide comprises a primer containing an oligo or poly dT sequence.
5 . The method of claim 4 wherein said oligo or poly dT sequence is at least about eight dT in length.
6 . The method of claim 1 wherein said random primer region comprises at least about six random nucleotides.
7 . The method of claim 6 wherein said random primer region comprises at least about nine random nucleotides.
8 . The method of claim 1 wherein said DNA polymerase activity is DNA dependent.
9 . The method of claim 8 wherein said DNA dependent polymerase activity is selected from exonuclease deficient Klenow, Taq polymerase activities, and combinations of exonuclease deficient Klenow and/or Taq polymerase activities.
10 . A method of amplifying RNA sequences complementary to, or present in, one or more than one target polynucleotide that is single stranded or made single stranded, comprising
a) forming double stranded cDNA templates containing sequences present in said target polynucleotide, wherein said sequences are operably linked to a promoter region, by
i) annealing said one or more than one single stranded target polynucleotide with a first oligonucleotide comprising a primer operably linked to a promoter region to form a first complex,
ii) synthesizing one or more than one first strand cDNA by reverse transcription of said first complex,
iii) degrading first oligonucleotides not used in i) or ii) above with exonuclease activity,
iv) annealing said one or more than one first stranded cDNA, after denaturing the hybrid(s) of single stranded target polynucleotide and cDNA or degrading the single stranded target polynucleotide from said hybrid(s), with a plurality of second oligonucleotides comprising a random primer region comprising at least about six random nucleotides to form a population of second complexes, and
v) forming one or more than one double stranded cDNA templates from said population of second complexes with DNA dependent DNA polymerase activity; and
b) transcribing said cDNA templates with an RNA polymerase capable of initiating transcription via said promoter region to produce amplified RNA (aRNA) containing sequences complementary to said one or more than one target polynucleotide;
c) forming additional double stranded DNA templates from said aRNA by
i) annealing said aRNA with a third oligonucleotide comprising a primer region operably linked to a promoter region to form a third complex,
ii) synthesizing the first strand of said additional DNA template by reverse transcription of said third complex to produce an aRNA/DNA hybrid,
iii) annealing said first strand of additional DNA template, after denaturing the aRNA/DNA hybrid or degrading the aRNA from said hybrid, with said first oligonucleotide to form a population of fourth complexes, and
iv) forming additional double stranded DNA templates from said population of fourth complexes with DNA dependent DNA polymerase activity; and
d) transcribing said additional DNA templates with an RNA polymerase capable of initiating transcription via the promoter region of said first oligonucleotide to produce amplified RNA (aRNA) containing sequences complementary to said target polynucleotide or via the promoter region of said third oligonucleotide to produce aRNA containing sequences present in said target polynucleotide.
11 . The method of claim 10 wherein said random primer region comprises at least about nine random nucleotides.
12 . The method of claim 10 wherein said formation of additional double stranded DNA templates from said aRNA further comprises degrading third oligonucleotides not used in c) i) or c) ii) with exonuclease activity before forming additional double stranded DNA templates.
13 . The method of claim 10 wherein said target polynucleotide is mRNA.
14 . The method of claim 10 wherein said more than one target polynucleotide are a cellular mRNA preparation.
15 . The method of claim 10 wherein said first oligonucleotide comprises a primer containing an oligo or poly dT sequence.
16 . The method of claim 15 wherein said oligo or poly dT sequence is at least about eight dT in length
17 . The method of claim 10 wherein said DNA dependent DNA polymerase activity is selected from exonuclease deficient Klenow, Taq polymerase activities, and combinations of exonuclease deficient Klenow and/or Taq polymerase activities.
18 . The method of claim 10 wherein said formation of additional double stranded DNA templates from said aRNA further comprises degrading third oligonucleotides not used in c) i) or c) ii) with exonuclease activity before forming additional double stranded DNA templates.
19 . The method of claim 10 wherein said third oligonucleotide comprises a known primer sequence or a random primer region of at least about six random nucleotides or at least about nine random nucleotides.
20 . The method of claim 10 wherein said first oligonucleotide comprises a T7 promoter region and said third oligonucleotide optionally comprises a T3 or SP6 promoter region.