ISOTHERMAL DNA AMPLIFICATION
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Abstract
The present invention provides for amplification of one or more polynucleotides by multi-staged linear amplifications using one or more RNA polymerases. At each stage RNA transcripts are accumulated at a linear rate, so that multiple stages provide for faster than linear transcript accumulation. In one aspect, the invention provides for polynucleotide amplification by ligating hairpin adaptors to an end of polynucleotides wherein the hairpin adaptors each contain a promoter sequence oriented so that transcription proceeds in the direction of the loop of the hairpin adaptor. Upon transcription through such loop region and to the complementary strand a replicate is made of the promoter sequence as well as the polynucleotide, thereby permitting exponential amplification upon reverse transcription, second strand synthesis, and repetition of the above cycle. Preferably such amplification is carried out under isothermal reaction conditions.
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Citations
34 Claims
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1-16. -16. (canceled)
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17. A polynucleotide comprising:
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a polynucleotide sequence; an adapter at one end of the polynucleotide sequence comprising a hairpin; and a promoter sequence positioned between the polynucleotide sequence and the hairpin, wherein the promoter sequence is oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30)
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- 29. A hairpin adapter comprising a loop region and a double stranded region, wherein the double stranded region comprises a promoter sequence oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin.
Specification