Isothermal DNA amplification

• US 20060166250A1
• Filed: 01/23/2006
• Published: 07/27/2006
• Est. Priority Date: 01/25/2005
• Status: Active Grant

• Current Assignee: Population Genetics Technologies Ltd.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Mikawa, Gi, Brenner, Sydney
• Granted Patent: US 7,579,153 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

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.

Claims

1. A method of amplifying a polynucleotide comprising the steps of:

providing a single stranded DNA containing a promoter sequence and a polynucleotide, the single stranded DNA having a 5′

end, a 3′

end, and complementary sequences at the 3′

end capable of forming a hairpin having a loop and a duplex, the promoter sequence being disposed between the polynucleotide and the 3′

end and the single stranded DNA being attached to a solid phase support by its 5′

end;

extending the duplex to form a double stranded DNA that includes the polynucleotide and complement thereof and the promoter sequence and complement thereof, thereby forming an operational promoter; and

generating RNA copies of the polynucleotide with an RNA polymerase that recognizes the operational promoter.

2. The method of claim 1 wherein said step of providing further includes providing a double strand DNA that contains in series an up-stream promoter, a first stem region, said loop region and complement thereof, a second stem region, said polynucleotide, and a primer binding site, wherein RNA copies of a strand of the double stranded DNA are generated by treating under synthesis conditions the double stranded DNA with an RNA polymerase that recognized the up-stream promoter, and wherein the RNA copies of a strand of the double stranded DNA are captured by specific hybridization of the primer binding site to a complement thereof attached to a solid phase support.

3. The method of claim 2 wherein said step of providing further includes treating under synthesis conditions said complement of said primer binding site with a reverse transcriptase to form said single stranded DNA.

4. The method of claim 3 said step of generating further including a step of converting said RNA copies into single stranded DNA by annealing a primer to said primer binding site and treating under synthesis conditions such primer with a reverse transcriptase.

5. The method according to claims 4 further including a step of converting to single stranded DNA said RNA copies of said polynucleotide by annealing a primer to said primer binding site and treating under synthesis conditions such primer with a reverse transcriptase.

6. A method of amplifying a strand of a polynucleotide, the method comprising the steps of:

ligating to an end of the polynucleotide a hairpin adaptor to form an augmented sequence, the hairpin adaptor having a loop region at one end and a duplex region containing a promoter sequence oriented so that transcription proceeds toward the loop region;

combining the augmented sequence, an RNA polymerase, a reverse transcriptase, a DNA polymerase, a first primer specific for the polynucleotide, and a helicase under reaction conditions such that the RNA polymerase generates transcripts that contain the promoter sequence, the first primers anneal to the transcripts and are extended by the reverse transcriptase to produce a heteroduplex containing a first complementary DNA strand, the heteroduplex is unwound at an end such that the loop region and the duplex region are regenerated in the first complementary DNA strand and the duplex region is extended by the DNA polymerase to reconstitute an augmented sequence, the RNA polymerase recognizing the promoter sequence of the reconstituted augmented sequence and synthesizing transcripts of the polynucleotide.

7. The method of claim 6 wherein said loop region contains from three to six nucleotides.

8. The method of claim 6 wherein said polynucleotide has an end distal to said end that said hairpin adaptor is ligated and wherein said distal end said polynucleotide has a primer binding site specific for said first primers.

9. A method of amplifying a polynucleotide in a reaction, the method comprising the steps of:

providing a first double stranded DNA having a hairpin at one end, the polynucleotide 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;

combining the double stranded DNA, an RNA polymerase, a reverse transcriptase, a DNA polymerase, a first primer specific for the polynucleotide, and a helicase under reaction conditions such that the RNA polymerase generates primary transcripts that contain the promoter sequence, the first primers anneal to the primary transcripts and are extended by the reverse transcriptase to produce a heteroduplex containing a complementary DNA strand, the heteroduplex is unwound at an end such that the hairpin is regenerated in the complementary DNA strand and is extended by the DNA polymerase to form an augmented sequence having a reconstituted promoter sequence, the RNA polymerase recognizing the reconstituted promoter sequence and synthesizing transcripts of the polynucleotide.

10. The method of claim 9 wherein said hairpin has a loop region that contains from three to six nucleotides.

11. The method of claim 10 wherein said polynucleotide has an end distal to said end that said hairpin and wherein said distal end said polynucleotide has a primer binding site specific for said first primers.

12. The method of claim 11 wherein said step of combining includes treating said heteroduplex by a helicase to unwind said heteroduplex.

13. A method of replicating a polynucleotide, the method comprising the steps of:

providing a double stranded DNA having a hairpin at one end, the polynucleotide 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;

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 the double stranded DNA containing a reconstituted promoter sequence, the RNA polymerase recognizing the reconstituted promoter sequence and synthesizing RNA transcripts.

14. The method of claim 13 wherein said step of generating includes forming a heteroduplex of said complementary DNA and said RNA transcript and wherein said step of displacing includes treating the heteroduplex with a helicase.

15. The method of claim 13 wherein said loop region contains from three to six nucleotides.

16. The method of claim 13 wherein said polynucleotide has an end distal to said end that said hairpin adaptor is ligated and wherein said distal end said polynucleotide has a primer binding site specific for said first primers.