Methods and reagents for combined PCR amplification and hybridization probing

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 deactivation step is performed.

We claim:  
     
         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 3′→5′ 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 3′→5′ 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 3′→5′ 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 3′→5′ 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 3′→5′ 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 3′→5′ 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 3′→5′ extension activity of a polymerase;  
 
   contacting the target nucleic acid sequence with an exonuclease activity inhibitor, the inhibitor being sufficient to inhibit the 3′→5′ 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 3′→5′ extension activity of a polymerase;  
 
   contacting the target nucleic acid sequence with an exonuclease activity inhibitor, the inhibitor being sufficient to inhibit the 3′→5′ 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 3′→5′ exonuclease activity of the polymerase; and    measuring the extent of fluorescence quenching of the oligonucleotide probe at the probe hybridization temperature.    
     
     
         20 . 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 the 5′→3′ exonuclease activity of a polymerase; and    a 3′ end which is rendered impervious to the 3′→5′ extension activity of a polymerase.    
     
     
         21 . The oligonucleotide probe of  claim 20  wherein said fluorescer molecule is a fluorescein dye and said quencher molecule is a rhodamine dye.