Methods and reagents for combined PCR amplification and hybridization probing
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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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Citations
32 Claims
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1-21. -21. (canceled)
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22. A method for performing combined PCR amplification and hybridization probing comprising the steps of:
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contacting a target nucleic acid sequence with PCR reagents, including at least two PCR primers and a polymerase enzyme, and an oligonucleotide probe comprising;
an oligonucleotide capable of hybridizing to a target polynucleotide sequence;
a fluorescer molecule attached to a first location on the oligonucleotide;
a quencher molecule attached to a second location on the oligonucleotide such that the quencher molecule substantially quenches the fluorescence of the fluorescer molecule whenever the oligonucleotide probe is not hybridized to the target polynucleotide sequence and such that the fluorescer molecule is substantially unquenched whenever the oligonucleotide probe is hybridized to the target polynucleotide sequence;
a 5′
end which is not recognized by a polymerase having a 5′
→
3′
exonuclease activity; and
a 3′
end which is not recognized by a polymerase having a 5′
→
3′
extension activity; and
subjecting the target nucleic sequence, the oligonucleotide probe, and the PCR reagents to thermal cycling, including a polymerization step, the thermal cycling being sufficient to amplify the target nucleic acid sequence specified by the PCR reagents. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification