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US 7,888,018 B2Granted
Methods and kits for nucleic acid amplification
AssigneeGenisphere, LLC
InventorsRobert C. Getts, Kelly Sensinger, James Kadushin
Life of this patent
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Abstract
Compositions and methods are provided for amplifying nucleic acid molecules. The nucleic acid molecules can be used in various research and diagnostic applications, such as gene expression studies involving nucleic acid microarrays.
Claims34
A method for synthesizing at least one asRNA molecule directly from at least one sRNA molecule, comprising:
- a) providing at least one sRNA molecule having a 5′ end and a 3′ end, said 3′ end comprising a first nucleotide sequence corresponding to the most 5′ end of the template strand of a consensus RNA polymerase recognition sequence and extending less than 19 nucleotides such that a complete template strand of a consensus RNA polymerase recognition sequence is not provided;
- b) annealing to the first nucleotide sequence of the 3′ end of said sRNA molecule a primer having a 5′ end and a 3′ end, said primer comprising a second nucleotide sequence corresponding to the most 5′ end of the non-template strand of said consensus RNA polymerase recognition sequence and being sufficient in length to anneal to said first nucleotide sequence;
- c) extending the 3′ end of said annealed primer such that a double stranded RNA/DNA duplex is formed, wherein the DNA strand of the RNA/DNA duplex comprises a third nucleotide sequence corresponding to a complete non-template strand of said consensus RNA polymerase recognition sequence at a 5′ end thereof;
- d) degrading at least the 3′ portion of the RNA strand of said double stranded
- RNA/DNA duplex, thereby providing at least a partially single stranded DNA molecule having a single stranded 5′ end, said single stranded 5′ end comprising the third nucleotide sequence corresponding to the complete non-template strand of said consensus RNA polymerase recognition sequence;
- e) synthesizing at least a partially double stranded DNA molecule from said at least partially single stranded DNA molecule such that said third nucleotide sequence corresponding to the complete non-template strand of said consensus RNA polymerase recognition sequence is converted into a double stranded consensus RNA polymerase promoter sequence;
- and
- f) initiating RNA transcription using an RNA polymerase which recognizes said double stranded consensus RNA polymerase promoter, thereby synthesizing at least one asRNA molecule directly from at least one sRNA molecule.
5 more elements
A method for synthesizing at least one cDNA molecule directly from at least one sRNA molecule, comprising:
- a) providing at least one sRNA molecule having a 5′ end and a 3′ end, said 3′ end comprising a first nucleotide sequence corresponding to the most 5′ end of the template strand of a consensus RNA polymerase recognition sequence and extending less than 19 nucleotides such that a complete template strand of a consensus RNA polymerase recognition sequence is not provided;
- b) annealing to the first nucleotide sequence of the 3′ end of said sRNA molecule a primer having a 5′ end and a 3′ end, said primer comprising a second nucleotide sequence corresponding to the most 5′ end of the non-template strand of said consensus RNA polymerase recognition sequence and being sufficient in length to anneal to said first nucleotide sequence;
- and c) extending the 3′ end of said annealed primer with reverse transcriptase, thereby synthesizing at least one cDNA molecule directly from at least one sRNA molecule, wherein the at least one cDNA molecule comprises a third nucleotide sequence corresponding to a complete non-template strand of said consensus RNA polymerase recognition sequence at a 5′ end thereof.
Dependencies are inferred from the claim text.