Methods for combined PCR amplification and hybridization probing using doubly labeled fluorescent probes
US5691146A · kind A · utility
Assignee
Inventor
Key dates
| Filing date | Sep 11, 1996 |
| Grant date | Nov 25, 1997 |
| Priority date | — |
| Expiry date | Sep 11, 2016 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY10S435/911
- WIPO fieldBiotechnology
- WIPO sectorChemistry
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.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.