Isothermal DNA amplification
First Claim
1. A method of replicating a polynucleotide sequence, the method comprising the steps of:
- providing a double stranded DNA having a hairpin at one end, the polynucleotide sequence at the other end, and disposed therebetween a promoter sequence oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin;
transcribing the double stranded DNA with an RNA polymerase that recognizes the promoter sequence to form an RNA transcript comprising copies of the promoter sequence and the polynucleotide sequence;
generating a complementary DNA from the RNA transcript;
displacing a 5'"'"' end of the RNA transcript from the complementary DNA so that the hairpin is reconstituted; and
extending the hairpin to generate double stranded DNA, wherein the generated double stranded DNA contains the reconstituted hairpin, a reconstituted promoter sequence and the polynucleotide sequence, thereby replicating the polynucleotide sequence.
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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.
150 Citations
16 Claims
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1. A method of replicating a polynucleotide sequence, the method comprising the steps of:
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providing a double stranded DNA having a hairpin at one end, the polynucleotide sequence at the other end, and disposed therebetween a promoter sequence oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin; transcribing the double stranded DNA with an RNA polymerase that recognizes the promoter sequence to form an RNA transcript comprising copies of the promoter sequence and the polynucleotide sequence; generating a complementary DNA from the RNA transcript; displacing a 5'"'"' end of the RNA transcript from the complementary DNA so that the hairpin is reconstituted; and extending the hairpin to generate double stranded DNA, wherein the generated double stranded DNA contains the reconstituted hairpin, a reconstituted promoter sequence and the polynucleotide sequence, thereby replicating the polynucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of replicating a polynucleotide sequence, the method comprising the steps of:
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providing a double stranded DNA having a hairpin at one end, the polynucleotide sequence at the other end, and disposed therebetween a promoter sequence oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin; transcribing the double stranded DNA with an RNA polymerase that recognizes the promoter sequence to form an RNA transcript comprising copies of the promoter sequence and the polynucleotide sequence, thereby replicating the polynucleotide sequence. - View Dependent Claims (11, 12, 13, 14, 15, 16)
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Specification