Methods and Reagents for Combined PCR Amplification

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.

1 - 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; 
   b) an oligonucleotide primer pair, wherein the oligonucleotide primer pair define the 5′ ends of two complementary strands of a double-stranded target sequence; and   c) a strand displacer.   
     
     
         23 . The kit of  claim 22  wherein said fluorescer molecule of said oligonucleotide 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  wherein said strand displacer is a helicase. 
     
     
         26 . The kit of  claim 22  further comprising a polymerase. 
     
     
         27 . The kit of  claim 26  wherein the polymerase is a thermostable DNA polymerase. 
     
     
         28 . The kit of  claim 22  further comprising at least four deoxyribonucleotide triphosphates (dNTPs). 
     
     
         29 . The kit of  claim 22  further comprising an aqueous buffer. 
     
     
         30 . The kit of  claim 22  further comprising a water soluble magnesium salt. 
     
     
         31 . The kit of  claim 22  further comprising a strand displacer inhibitor. 
     
     
         32 . 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; 
   b) an oligonucleotide primer pair, wherein the oligonucleotide primer pair define the 5′ ends of two complementary strands of a double-stranded target sequence; and   c) a strand displacer.   
     
     
         33 . The kit of  claim 32  wherein said fluorescer molecule of said oligonucleotide probe is a fluorescein dye and said quencher molecule of said oligonucleotide probe is a rhodamine dye. 
     
     
         34 . The kit of  claim 32  wherein said strand displacer is a helicase. 
     
     
         35 . The kit of  claim 32  further comprising a polymerase. 
     
     
         36 . The kit of  claim 35  wherein the polymerase is a thermostable DNA polymerase. 
     
     
         37 . The kit of  claim 32  further comprising at least four deoxyribonucleotide triphosphates (dNTPs). 
     
     
         38 . The kit of  claim 32  further comprising an aqueous buffer. 
     
     
         39 . The kit of  claim 32  further comprising a water soluble magnesium salt. 
     
     
         40 . The kit of  claim 32  further comprising a strand displacer inhibitor.