Methods for combined PCR amplification and hybridization probing

Details Methods for combined PCR amplification and hybridization probing Methods for combined PCR amplification and hybridization probing

1996-09-11

1997-11-25

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 5, 435 911, 435 912, 536 221, 536 231, 536 243, 536 2431, 536 2432, 536 2433, 536 245, C12Q 168, C12Q 170, C12P 1934, C07H 2104

Patent

active

056911467

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'.fwdarw.3' exonuclease activity of a polymerase and the 3'.fwdarw.5' 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'.fwdarw.3' exonuclease activity, (ii) a 5'.fwdarw.3' exonuclease inhibitor is included, and (iii) an exonuclease reactivation step is performed.

REFERENCES:
patent: 5210015 (1993-05-01), Gelfand et al.
Sixou et al, "Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET)", Nucleic Acids Res. 22(4):662-668, 1994.

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