US5691146A
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US
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Methods for combined PCR amplification and hybridization probing using doubly labeled fluorescent probes
Assignee: Applied Biosystems LLC
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Filing: 1996-09-11
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Granted: 1997-11-25
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Expires: 2016-09-11
Abstract
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 3'->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'->3' exonuclease activity, (ii) a 5'->3' exonuclease inhibitor is included, and (iii) an exonuclease reactivation step is performed.
Inventors
- 👤 Paul E. Mayrand
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 14:47
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C07; C07H; C07H21/00; C07H21/04; C12Q1/6844; C12Q1/6853; C12Q1/686; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/8215; Y10S435/911
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
176 words
A method for performing combined PCR amplification and hybridization probing comprising the steps of: contacting a target nucleic acid 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 nucleic acid; a fluorescer molecule attached to a first end of the oligonucleotide; a quencher molecule attached to a second end of the oligonucleotide such that the quencher molecule substantially quenches the fluorescer molecule whenever the oligonucleotide probe is in a single-stranded state and such that the fluorescer is substantially unquenched whenever the oligonucleotide probe is hybridized to the target nucleic acid; a 5' end which is rendered impervious to digestion by the 5'→3' exonuclease activity of a polymerase; and a 3' end which is rendered impervious to the 5'→3' extension activity of the polymerase; and subjecting the target nucleic acid, 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 specified by the PCR reagents.
Claim 2
Independent Claim
176 words
A method for performing combined PCR amplification and hybridization probing comprising the steps of: contacting a target nucleic acid 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 nucleic acid; a fluorescer molecule attached to a first end of the oligonucleotide; a quencher molecule attached to a second end of the oligonucleotide such that the quencher molecule substantially quenches the fluorescer molecule whenever the oligonucleotide probe is in a single-stranded state and such that the fluorescer is substantially unquenched whenever the oligonucleotide probe is hybridized to the target nucleic acid; a 5' end which is rendered impervious to digestion by the 5'→3' exonuclease activity of a polymerase; and a 3' end which is rendered impervious to the 5'→3' extension activity of the polymerase; and subjecting the target nucleic acid, 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 specified by the PCR reagents.
Claim 3
Dependent Claim
33 words
The method of claim 1 further comprising the step of measuring the extent of fluorescence quenching of the oligonucleotide probe, such measurement being performed subsequent to thermocycling and at a probe hybridization temperature.
Claim 4
Dependent Claim
19 words
The method of claim 1 wherein the target nucleic acid sequence is located within one or more fixed cells.
Claim 5
Dependent Claim
43 words
The method of claim 3 further comprising the step of measuring the extent of fluorescence quenching of the oligonucleotide probe at a probe hybridization temperature in a manner which locates the probe within the individual cells originally containing the target nucleic acid sequence.
Claim 6
Dependent Claim
22 words
The method of claim 3 wherein the fixed cells, the PCR reagents, and the oligonucleotide probe are located in a containment assembly.
Claim 7
Dependent Claim
25 words
The method of claim 2 wherein the probe hybridization temperature is less than or equal to the temperature of the polymerization step of the thermocycling.
Claim 8
Dependent Claim
44 words
The method of claim 2 further comprising the step of adding a strand displacer prior to thermal cycling for preventing the oligonucleotide probe from blocking the 5'→3' extension of an upstream PCR primer during the polymerization step while not substantially displacing the PCR primer.
Claim 9
Dependent Claim
25 words
The method of claim 7 further comprising a strand displacer deactivation step subsequent to thermocycling for deactivating the strand displacement activity of the strand displacer.
Claim 10
Dependent Claim
33 words
The method of claim 1 further comprising the step of adding a strand displacer prior to thermal cycling for preventing the oligonucleotide probe from blocking the 5'→3' extension of an upstream PCR primer.
Claim 11
Dependent Claim
12 words
The method of claim 9 wherein the strand displacer is a helicase.
References Cited (Backward Citations)
US5210015A
Cited
WO1993010267A1
Cited
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