Global linear non-biased nucleic acid amplification
First Claim
1. A method of preparing amplified RNA sequences 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, by i) annealing said single stranded target polynucleotide with a plurality of first oligonucleotides, each comprising a random primer sequence, to form a first complex, ii) synthesizing a first strand cDNA by reverse transcription of said first complex and adding a homopolymer tail to said first strand cDNA by use of terminal deoxyribonucleotidyl transferase activity, iii) optionally degrading first oligonucleotides not used in i) or ii) above with exonuclease activity, iv) annealing said first strand cDNA, after denaturing the mRNA/cDNA hybrid or degrading the RNA from said hybrid, with a second oligonucleotide comprising a primer sequence, complementary to said homopolymer tail and operably linked to a promoter region, to form a population of second complexes, and v) forming double stranded cDNA templates from said population of second complexes with 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 of said target polynucleotide.
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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.
14 Citations
33 Claims
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1. A method of preparing amplified RNA sequences 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, by i) annealing said single stranded target polynucleotide with a plurality of first oligonucleotides, each comprising a random primer sequence, to form a first complex, ii) synthesizing a first strand cDNA by reverse transcription of said first complex and adding a homopolymer tail to said first strand cDNA by use of terminal deoxyribonucleotidyl transferase activity, iii) optionally degrading first oligonucleotides not used in i) or ii) above with exonuclease activity, iv) annealing said first strand cDNA, after denaturing the mRNA/cDNA hybrid or degrading the RNA from said hybrid, with a second oligonucleotide comprising a primer sequence, complementary to said homopolymer tail and operably linked to a promoter region, to form a population of second complexes, and v) forming double stranded cDNA templates from said population of second complexes with 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 of said target polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 31)
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17. 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 single stranded target polynucleotide with a plurality of first oligonucleotides, each comprising a random primer sequence, to form a first complex, ii) synthesizing a first strand cDNA by reverse transcription of said first complex and adding a homopolymer tail to said first strand cDNA by use of terminal deoxyribonucleotidyl transferase activity, iii) optionally degrading first oligonucleotides not used in i) or ii) above with exonuclease activity, iv) annealing said first strand cDNA, after denaturing the mRNA/cDNA hybrid or degrading the RNA from said hybrid, with a second oligonucleotide comprising a primer sequence, complementary to said homopolymer tail and operably linked to a promoter region, to form a population of second complexes, and v) forming 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 target polynucleotide;
c) forming additional double stranded DNA templates from said aRNA by i) annealing said aRNA with a third oligonucleotide comprising a primer sequence 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, iii) annealing said first strand of additional DNA template, after denaturing the aRNA/DNA hybrid or degrading the aRNA from said hybrid, with said second 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 third oligonucleotide to produce amplified RNA (aRNA) containing sequences complementary to said target polynucleotide or via the promoter region of said second oligonucleotide to produce aRNA containing sequences of said target polynucleotide. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33)
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Specification