Methods and compositions for discrete melt analysis

Abstract

Methods and reagent for determining the presence and/or for quantifying the amount of a target nucleic acid sequences in a sample are provided. In some aspects, the methods comprise performing a melt analysis by detecting, a signal from a probe at a temperature that is lower than the Tm of the probe and a signal at a temperature that is higher than the Tm of the probe, without detecting a signal at the Tm of the probe.

What is claimed is: 
     
         1 . A method of detecting the presence of a first and second target nucleic acid in a sample, the method comprising the steps of:
 a) forming an amplification reaction mixture comprising a first and second fluorescent signal-generating target-specific probe and the sample potentially containing the first and second target nucleic acid;   b) partitioning the reaction mixture so that each partition contains, on average, zero or one of the first and/or second target nucleic acids;   c) acquiring a first, second and third set of fluorescent intensity values from images of at least a subset of partitions at preselected temperatures T1, T2 and T3 and for each partition in the subset of partitions, determining a first fluorescent intensity value at the preselected temperature T1, a second fluorescent intensity value at the preselected temperature T2 and a third fluorescent intensity value at the preselected temperature T3;   d) subjecting the partitions containing the amplification reaction mixture to conditions for amplification of the first and second target nucleic acids and modification of the first signal generating probe in the presence of its target nucleic acid to form a first duplex nucleic acid having a first predetermined Tm, and modification of the second signal generating probe in the presence of its target nucleic acid to form a second duplex nucleic acid having a second predetermined Tm;   e) acquiring a fourth set of fluorescent intensity values from images of the subset of partitions at the temperature T1 which is lower than the first predetermined Tm and calculating a fourth intensity value for each partition;   f) acquiring a fifth set of fluorescent intensity values from images of the subset of partitions at the temperature T2 that is higher than the first predetermined Tm but lower than the second predetermined Tm, and calculating a fifth intensity value for each partition;   g) acquiring a sixth set of fluorescent intensity values from images of the subset of partitions at the temperature T3 that is higher than the second predetermined Tm and calculating a sixth intensity value for each partition;   h) for each partition, dividing the fourth intensity value by the first intensity value to obtain a seventh intensity value associated with T1;   i) for each partition, dividing the fifth intensity value by the second intensity value to obtain a eighth intensity value associated with T2;   j) for each partition, dividing the sixth intensity value by the third intensity value to obtain a ninth intensity value associated with T3;   k) for each partition, detecting a change between the eighth intensity value and the seventh intensity value to determine the presence of the first target nucleic; and   l) for each partition, detecting a change between the ninth average intensity value and the eighth average intensity value to determine the presence of second target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the first and second fluorescent signal-generating probes have the same signal-generating label. 
     
     
         3 . The method of  claim 2 , wherein the signal generating label is a first member of a reporter-quencher pair and the first and second duplex nucleic acids comprise a second member of the reporter-quencher pair. 
     
     
         4 . The method of  claim 3 , wherein the first and second signal-generating probes comprise a first non-natural nucleotide to which the first member of the reporter-quencher pair is attached, and the first and second duplex nucleic acids comprise a second non-natural nucleotide to which the second member of the reporter-quencher pair is attached, and the first and second non-natural nucleotides are capable of base-pairing with each other. 
     
     
         5 . The method of  claim 4 , wherein the non-natural nucleotides are one of iso-C or iso-G. 
     
     
         6 . The method of  claim 1 , wherein the first and second signal-generating probes are cleavable probes that are cleaved during amplification. 
     
     
         7 . The method of  claim 1 , wherein the change between the eighth intensity value and the seventh intensity value exceeds a predetermined threshold, and wherein the change between the ninth intensity value and the eighth intensity value exceeds a predetermined threshold. 
     
     
         8 . A method of detecting the presence of a target nucleic acid in a sample, the method comprising the steps of:
 a) forming an amplification reaction mixture comprising a fluorescent signal-generating target-specific probe and the sample containing the target nucleic acid;   b) partitioning the reaction mixture so that each partition contains, on average, zero or one of the target nucleic acid;   c) acquiring a first and second set of fluorescent intensity values from images of at least a subset of partitions at preselected temperatures T1 and T2, and for each partition in the subset of partitions, determining a first fluorescent intensity value at the preselected temperature T1 and a second fluorescent intensity value at the preselected temperature T2;   d) subjecting the partitions containing the amplification reaction mixture to conditions for amplification of the target nucleic acids and modification of the signal generating probe in the presence of its target nucleic acid to form a duplex nucleic acid having a predetermined Tm;   e) acquiring a third set of fluorescent intensity values from images of the subset of partitions at the temperature T1 which is lower than the predetermined Tm and calculating a third intensity value for each partition;   f) acquiring a fourth set of fluorescent intensity values from images of the subset of partitions at the temperature T2 that is higher than the predetermined Tm, and calculating a fourth intensity value for each partition;   g) for each partition, dividing the third intensity value by the first intensity value to obtain a fifth intensity value associated with T1;   h) for each partition, dividing the fourth intensity value by the second intensity value to obtain a sixth intensity value associated with T2; and   i) for each partition, detecting a change between the sixth intensity value and the fifth intensity value to determine the presence of the target nucleic acid.   
     
     
         9 . The method of  claim 8 , wherein the target nucleic acid is a first target nucleic acid, the signal generating target-specific probe is a first fluorescent signal-generating target-specific probe and the predetermined Tm is a first predetermined Tm, further comprising detecting the presence of a second target nucleic acid, wherein the reaction mixture further comprises a second fluorescent signal-generating target-specific probe, the method further comprising the steps of:
 j) in step (b), partitioning the reaction mixture so that each partition contains, on average, zero or one of the first and/or second target nucleic acid;   k) in step (c), acquiring a fifth set of fluorescent intensity values from images of at least a subset of partitions at a preselected temperature T3, and for each partition in the subset of partitions, determining a seventh fluorescent intensity value at the preselected temperature T3;   l) in step (d), subjecting the partitions containing the amplification reaction mixture to conditions for amplification of the target nucleic acids and modification of the second signal generating probe in the presence of its target nucleic acids to form a second duplex nucleic acid having a second predetermined Tm;   m) acquiring a sixth set of fluorescent intensity values from images of the subset of partitions at the temperature T3 that is higher than the second predetermined Tm and calculating an eighth intensity value for each partition;   n) for each partition, dividing the eighth intensity value by the seventh intensity value to obtain a ninth intensity value associated with T3; and   o) for each partition, detecting a change between the ninth intensity value with the sixth intensity value to determine the presence of the second target nucleic acid.   
     
     
         10 . The method of  claim 8 , wherein the change between the sixth and fifth intensity values and the change between the ninth and the sixth intensity values exceeds a predetermined threshold. 
     
     
         11 . The method of  claim 9 , wherein the change between the sixth and fifth intensity values and the change between the ninth and the sixth intensity values exceeds a predetermined threshold. 
     
     
         12 . The method of  claim 9 , wherein the first and second fluorescent signal-generating probes have the same signal-generating label. 
     
     
         13 . The method of  claim 9 , wherein the signal generating label is a first member of a reporter-quencher pair and the first and second duplex nucleic acids comprise a second member of the reporter-quencher pair. 
     
     
         14 . The method of  claim 9 , wherein the first and second signal-generating probes comprise a first non-natural nucleotide to which the first member of the reporter-quencher pair is attached, and the first and second duplex nucleic acids comprise a second non-natural nucleotide to which the second member of the reporter-quencher pair is attached, and the first and second non-natural nucleotides are capable of base-pairing with each other. 
     
     
         15 . The method of  claim 14 , wherein the non-natural nucleotides are one of iso-C or iso-G. 
     
     
         16 . The method of  claim 9 , wherein the first and second signal-generating probes are cleavable probes that are cleaved during amplification.