Title:
PCR増幅とハイブリダイゼーションプロービングアッセイを組み合わせた方法およびそのための試薬
Document Type and Number:
Japanese Patent JP4040519
Kind Code:
B2
Abstract:
An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5′ ends protected, wherein (i) the polymerase lacks 5′→3′ exonuclease activity, (ii) a 5′→3′ exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.
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Inventors:
Paul E. Mayland
Application Number:
JP2003099758A
Publication Date:
January 30, 2008
Filing Date:
April 02, 2003
Export Citation:
Assignee:
Appraera Corporation
International Classes:
C12N15/09; C07H21/04; C12P19/34; C12Q1/68; G01N21/64; G01N21/77; G01N21/78; G01N33/53; G01N33/566
Domestic Patent References:
| JP723800A |
Foreign References:
| EP0601889A1 | ||||
| WO1994016090A1 |
Other References:
Nucleic Acids Res.,Vol.22,No.12(1994)p.2424-2425
Biochemistry,Vol.34,No.1(1995.Jan.)p.285-292
Biochemistry,Vol.34,No.1(1995.Jan.)p.285-292
Attorney, Agent or Firm:
Hidesaku Yamamoto