Methods and reagents for combined PCR amplification and hybridization probing

• US 7,241,596 B2
• Filed: 09/20/2004
• Issued: 07/10/2007
• Est. Priority Date: 05/05/1995
• Status: Expired due to Fees

• Current Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Mayrand, Paul E.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/946,458
• Publication Number:
  US 20050227247A1
• Time in Patent Office: 1,023 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/24.5, 514/44
• US Class Current: 435/91.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/911 using fungi

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.

Claims

1. A method for performing combined PCR amplification and hybridization probing comprising the steps of:

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.

2. The method of claim 1 wherein said fluorescer molecule is a flourescein dye and said quencher molecule is a rhodamine dye.

3. The method of claim 2 wherein said first location on said oligonucleotide is the 5′

end.

4. 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.

5. The method of claim 1 wherein the target nucleic acid sequence is located within one or more fixed cells.

6. The method of claim 5 further comprising the step of measuring the extent of fluorscence quenching of the oligonucleotide probe at a hybridization temperature in a manner which locates the probe within the individual cells originally containing the target nucleic acid sequence.

7. The method of claim 5 wherein the fixed cells, the PCR reagents,and the oligonucleotide probe are located in a containment assembly.

8. The method of claim 1 wherein the probe hybridization temperature is less than or equal to the temperature of the polymerization step of the thermocycling.

9. 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 during the polymerization step.

10. The method of claim 9 wherein the strand displacer is a helicase.

11. The method of claim 1 wherein the fluorescer and quencher are separated by at least 18 nucleotides.