Methods and reagents for combined PCR amplification

• US 7,847,076 B2
• Filed: 08/18/2008
• Issued: 12/07/2010
• Est. Priority Date: 05/05/1995
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Mayrand, Paul E.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/193,667
• Publication Number:
  US 20090258351A1
• Time in Patent Office: 841 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/26.6, 435/6, 435/91.1, 435/91.2
• US Class Current: 536/23.1
• 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. An oligonucleotide probe comprising:

an oligonucleotide capable of hybridizing to a target polynucleotide sequence;

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 fluorescence of 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 in a double-stranded state;

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 a polymerase.

2. The oligonucleotide probe of claim 1 wherein said fluorescer molecule is a fluorescein dye and said quencher molecule is a rhodamine dye.

3. The oligonucleotide probe of claim 2 wherein said first end of said oligonucleotide is the 5′

end.

4. 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 end of the oligonucleotide;

a quencher molecule attached to a second end of the oligonucleotide such that quencher molecule substantially quenches the fluorescence of 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 in a double-stranded state;

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 a polymerase; 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.

5. The method of claim 4 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.

6. The method of claim 4 wherein the target nucleic acid sequence is located within one or more fixed cells.

7. The method of claim 6 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.

8. The method of claim 6 wherein the fixed cells, the PCR reagents, and the oligonucleotide probe are located in a containment assembly.

9. The method of claim 5 wherein the probe hybridization temperature is less than or equal to the temperature of the polymerization step of the thermocycling.

10. The method of claim 5 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.

11. The method of claim 10 wherein the strand displacer lacks strand displacement activity at the hybridization temperature.

12. The method of claim 10 further comprising the step of contacting the target nucleic acid sequence with a strand displacer inhibitor prior to thermocycling, the inhibitor being sufficient to inhibit the strand displacement activity of the strand displacer at the hybridization temperature.

13. The method of claim 10 further comprising a strand displacer deactivation step subsequent to thermocycling for deactivating the strand displacement activity of the strand displacer.

14. The method of claim 4 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.

15. The method of claim 14 wherein the strand displacer is a helicase.

16. 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 substantially lacking any 5′

→

3′

exonuclease activity, and an oligonucleotide probe comprising;

an oligonucleotide;

a fluorescer molecule attached to a first end of the oligonucleotide;

a quencher molecule attached to a second end of the oligonucleotide such that quencher molecule substantially quenches the fluorescence of 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 in a double-stranded state; and

a 3′

end which is rendered impervious to the 5′

→

3′

extension activity of a polymerase; and

subjecting the target nucleic sequence, the oligonucleotide probe, and the PCR reagents to thermal cycling sufficient to amplify the target nucleic acid sequence specified by the PCR reagents.

17. The method of claim 16 further comprising the step of measuring the extent of fluorescence quenching of the oligonucleotide probe at a probe hybridization temperature.

18. 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;

a fluorescer molecule attached to a first end of the oligonucleotide;

a quencher molecule attached to a second end of the oligonucleotide such that quencher molecule substantially quenches the fluorescence of 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 in a double-stranded state; and

a 3′

end which is rendered impervious to the 5′

→

3′

extension activity of a polymerase;

contacting the target nucleic acid sequence with an exonuclease activity inhibitor, the inhibitor being sufficient to inhibit the 5′

→

3′

exonuclease activity of the polymerase at a probe hybridization temperature;

subjecting the target nucleic sequence, the oligonucleotide probe, the PCR reagents, and the inhibitor to thermal cycling sufficient to amplify the target nucleic acid sequence specified by the PCR reagents; and

measuring the extent of fluorescence quenching of the oligonucleotide probe at the probe hybridization temperature.

19. 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;

a fluorescer molecule attached to a first end of the oligonucleotide;

a quencher molecule attached to a second end of the oligonucleotide such that quencher molecule substantially quenches the fluorescence of 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 in a double-stranded state; and

a 3′

end which is rendered impervious to the 5′

→

3′

extension activity of a polymerase;

contacting the target nucleic acid sequence with an exonuclease activity inhibitor, the inhibitor being sufficient to inhibit the 5′

→

3′

exonuclease activity of the polymerase at a probe hybridization temperature;

subjecting the target nucleic sequence, the oligonucleotide probe, and the PCR reagents to thermal cycling sufficient to amplify the target nucleic acid sequence specified by the PCR reagents;

deactivating the 5′

→

3′

exonuclease activity of the polymerase; and

measuring the extent of fluorescence quenching of the oligonucleotide probe at the probe hybridization temperature.