Methods and Reagents for Combined PCR Amplification

• US 20080050742A1
• Filed: 07/09/2007
• Published: 02/28/2008
• Est. Priority Date: 05/05/1995
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Mayrand, Paul
• Granted Patent: US 7,413,708 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-21. -21. (canceled)

22. A kit for performing a combined PCR amplification and hybridization probing comprising:

(a) 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 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 rendered impervious to digestion by a 5′

to 3′

exonuclease activity of a polymerase; and

a 3′

end which is rendered impervious to a 5′

to 3′

extension activity of the polymerase,

and,

(b) an oligonucleotide primer pair, wherein the oligonucleotide primer pair define the 5′

ends of two complementary strands of a double-stranded target sequence.

23. The kit of claim 22 wherein said fluorescer molecule of said oligonuceotide probe is a fluorescein dye and said quencher molecule of said oligonucleotide probe is a rhodamine dye.

24. The kit of claim 22 wherein said first location of said oligonucleotide of the oligonucleotide probe is the 5′

end.

25. The kit of claim 22 further comprising a polymerase.

26. The kit of claim 25 wherein the polymerase is a thermostabile DNA polymerase.

27. The kit of claim 22 further comprising at least four deoxyribonucleotide triphosphates (dNTPs).

28. The kit of claim 22 further comprising an aqueous buffer.

29. The kit of claim 22 further comprising a water soluble magnesium salt.

30. A kit for performing a combined PCR amplification and hybridization probing comprising:

(a) 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 first location and the second location are separated by at least 18 nucleotides;

a 5′

end which is rendered impervious to digestion by a 5′

to 3′

exonuclease activity of a polymerase; and

a 3′

end which is rendered impervious to a 5′

to 3′

extension activity of the polymerase,

and,

(b) an oligonucleotide primer pair, wherein the oligonucleotide primer pair define the 5′

ends of two complementary strands of a double-stranded target sequence.

31. The kit of claim 30 wherein said fluorescer molecule of said oligonucleotide probe is a fluorescein dye and said quencher molecule of said oligonucleotide probe is a rhodamine dye.

32. The kit of claim 30 further comprising a polymerase.

33. The kit of claim 32 wherein the polymerase is a thermostabile DNA polymerase.

34. The kit of claim 30 further comprising at least four deoxyribonucleotide triphosphates (dNTPs).

35. The kit of claim 30 further comprising an aqueous buffer.

36. The kit of claim 30 further comprising a water soluble magnesium salt.