Detection of target nucleic acid sequences using different detection temperatures

A method for SNP genotyping of a nucleic acid sequence in a sample using different detection temperatures, comprising:

• (a) incubating the sample comprising the nucleic acid sequence containing a SNP (single nucleotide polymorphism) site and a signal-generating means for detection of SNP alleles in a single reaction vessel and detecting a generated signal by using a single type of detector;
• wherein each of the SNP alleles is detected by a corresponding signal-generating means;
• wherein one of the SNP alleles has a relatively high detection temperature and the other has a relatively low detection temperature determined by the corresponding signal-generating means;
• Wherein the relatively high detection temperature is a temperature capable of generating a signal for the SNP allele having the relatively high detection temperature and the relatively low detection temperature is a temperature capable of generating both of a signal for the SNP allele having the relatively low detection temperature and a signal for the SNP allele having the relatively high detection temperature;
• wherein signals to be generated by the signal-generating means are not differentiated by the single type of detector;
• wherein the detection is performed at both the relatively high detection temperature and the relatively low detection temperature;
• (b) determining a SNP genotype by a difference between the signal detected at the relatively high detection temperature and the signal detected at the relatively low detection temperature in the step (a).

A method for detecting at least three target nucleic acid sequences in a sample using different detection temperatures, comprising:

• (a) incubating the sample with at least three signal-generating means for detection of the at least three target nucleic acid sequences in a single reaction vessel and detecting a generated signal by using a single type of detector;
• wherein each of the at least three target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein each of the at least three target nucleic acid sequences has a different detection temperature determined by the corresponding signal-generating means;
• wherein a detection temperature is a temperature capable of generating not only a signal for a target nucleic acid sequence having the detection temperature but also a signal for a target nucleic acid sequence having a higher detection temperature than the detection temperature;
• wherein signals to be generated by the signal-generating means are not differentiated by the single type of detector;
• wherein the detection is performed at each of the different detection temperatures;
• and
• (b) determining the presence of the at least three target nucleic acid sequences by the signals detected in the step (a);
• wherein when the presence of a target nucleic acid sequence having a certain detection temperature among the at least three target nucleic acid sequences is determined, the presence of the target nucleic acid sequence having the certain detection temperature is determined by a difference between the signal detected at one or more detection temperatures higher than the certain detection temperature and the signal detected at the certain detection temperature;
• wherein when the certain detection temperature is a relatively highest detection temperature among the detection temperatures, the presence of a target nucleic acid sequence is determined by the signal detected at the certain detection temperature.

A method for detecting two target nucleic acid sequences in a sample using different detection temperatures and melting analysis, comprising:

• (a) incubating the sample with two signal-generating means for detection of the two target nucleic acid sequences in a single reaction vessel and detecting a generated signal by using a single type of detector;
• wherein each of the target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein one of the two target nucleic acid sequences has a relatively high detection temperature and the other has a relatively low detection temperature determined by the corresponding signal-generating means;
• wherein the relatively high detection temperature is a temperature capable of generating a signal for the target nucleic acid sequence having the relatively high detection temperature and the relatively low detection temperature is a temperature capable of generating both of a signal for the target nucleic acid sequence having the relatively low detection temperature and a signal for the target nucleic acid sequence having the relatively high detection temperature;
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• wherein the detection is performed at the relatively high detection temperature;
• (b) performing a melting analysis of the incubation resultant of the step (a) over a range of temperatures for determining the presence of the target nucleic acid sequence having the relatively low detection temperature;
• and
• (c) determining the presence of the target nucleic acid sequence having the relatively high detection temperature by the signal detected in the step (a) and the presence of the target nucleic acid sequence having the relatively low detection temperature by using the result of the melting analysis in the step (b).

A method for detecting at least three target nucleic acid sequences in a sample using detection temperature analysis and melting analysis, comprising:

• (a) incubating the sample with at least three signal-generating means for detection of the at least three target nucleic acid sequences in a single reaction vessel and detecting a generated signal by using a single type of detector;
• wherein each of the at least three target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein each of the at least three target nucleic acid sequences has a different detection temperature determined by the corresponding signal-generating means;
• wherein a detection temperature is a temperature capable of generating not only a signal for a target nucleic acid sequence having the detection temperature but also a signal for a target nucleic acid sequence having a higher detection temperature than the detection temperature;
• wherein signals to be generated by the signal-generating means are not differentiated by the single type of detector;
• wherein some of the at least three target nucleic acid sequences are detected by detection temperature analysis and the detection is performed at both the detection temperature of said some of the at least three target nucleic acid sequences and one or more detection temperatures higher than the detection temperatures;
• (b) performing a melting analysis of the incubation resultant of the step (a) over a range of temperatures for determining the presence of the other target nucleic acid sequences than said some of the at least three the target nucleic acid sequences;
• and
• (c) determining (i) the presence of said some of the target nucleic acid sequences by the signal detected in the step (a);
• wherein when the presence of a target nucleic acid sequence having a certain detection temperature among said some of the at least three target nucleic acid sequences is determined, the presence of the target nucleic acid sequence having the certain detection temperature is determined by a difference between the signal detected at one or more detection temperatures higher than the certain detection temperature and the signal detected at the certain detection temperature;
• wherein when the certain detection temperature is a relatively highest detection temperature among the detection temperatures, the presence of the target nucleic acid sequence is determined by the signal detected at the certain detection temperature;
• and (ii) the presence of the other target nucleic acid sequences than said some of the at least three target nucleic acid sequences is determined by the result of the melting analysis in the step (b).

A computer readable storage medium containing instructions to configure a processor to perform a method for determining the presence of two target nucleic acid sequences in a sample using different detection temperatures, the method comprising:

• (a) receiving both a signal detected at a relatively high detection temperature and a signal detected at a relatively low detection temperature, wherein each of the target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein a generated signal is detected by using a single type of detector;
• wherein one of the two target nucleic acid sequences has the relatively high detection temperature and the other has the relatively low detection temperature determined by the corresponding signal-generating means;
• wherein the relatively high detection temperature is a temperature capable of generating a signal for the target nucleic acid sequence having the relatively high detection temperature and the relatively low detection temperature is a temperature capable of generating both of a signal for the target nucleic acid sequence having the relatively low detection temperature and a signal for the target nucleic acid sequence having the relatively high detection temperature;
• and
• (b) determining the presence of the two target nucleic acid sequences by the signal received;
• wherein (i) the presence of the target nucleic acid sequence having the relatively high detection temperature is determined by the signal detected at the relatively high detection temperature and (ii) the presence of the target nucleic acid sequence having the relatively low detection temperature is determined by a difference between the signal detected at the relatively high detection temperature and the signal detected at the relatively low detection temperature.

A computer program to be stored on a computer readable storage medium to configure a processor to perform a method for determining the presence of two target nucleic acid sequences in a sample, the method comprising:

• (a) receiving both a signal detected at a relatively high detection temperature and a signal detected at a relatively low detection temperature, wherein each of the target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein a generated signal is detected by using a single type of detector;
• wherein one of the two target nucleic acid sequences has the relatively high detection temperature and the other has the relatively low detection temperature determined by the corresponding signal-generating means;
• wherein the relatively high detection temperature is a temperature capable of generating a signal for the target nucleic acid sequence having the relatively high detection temperature and the relatively low detection temperature is a temperature capable of generating both of a signal for the target nucleic acid sequence having the relatively low detection temperature and a signal for the target nucleic acid sequence having the relatively high detection temperature;
• and
• (b) determining the presence of the two target nucleic acid sequences by the signal received;
• wherein (i) the presence of the target nucleic acid sequence having the relatively high detection temperature is determined by the signal detected at the relatively high detection temperature and (ii) the presence of the target nucleic acid sequence having the relatively low detection temperature is determined by a difference between the signal detected at the relatively high detection temperature and the signal detected at the relatively low detection temperature.

A computer readable storage medium containing instructions to configure a processor to perform a method for SNP genotyping of a nucleic acid sequence in a sample using different detection temperatures, the method comprising:

• (a) receiving both a signal detected at a relatively high detection temperature and a signal detected at a relatively low detection temperature, wherein each of the SNP alleles is detected by a corresponding signal-generating means;
• wherein one of the SNP alleles has a relatively high detection temperature and the other has a relatively low detection temperature determined by the corresponding signal-generating means;
• wherein the relatively high detection temperature is a temperature capable of generating a signal for the SNP allele having the relatively high detection temperature and the relatively low detection temperature is a temperature capable of generating both of a signal for the SNP allele having the relatively low detection temperature and a signal for the SNP allele having the relatively high detection temperature;
• (b) determining a SNP genotype by a difference between the signals received.

A computer readable storage medium containing instructions to configure a processor to perform a method for determining the presence of at least three target nucleic acid sequences in a sample using different detection temperatures, the method comprising:

• (a) receiving signals detected at at least three detection temperatures;
• wherein each of the at least three target nucleic acid sequences is detected by a corresponding signal-generating means;
• wherein each of the at least three target nucleic acid sequences has a different detection temperature determined by the corresponding signal-generating means;
• wherein a detection temperature is a temperature capable of generating not only a signal for a target nucleic acid sequence having the detection temperature but also a signal for a target nucleic acid sequence having a higher detection temperature than the detection temperature;
• wherein signals to be generated by the signal-generating means are not differentiated by the single type of detector;
• wherein the detection is performed at each of the different detection temperatures;
• and
• (b) determining the presence of the at least three target nucleic acid sequences by the signal received;
• wherein when the presence of a target nucleic acid sequence having a certain detection temperature among the at least three target nucleic acid sequences is determined, the presence of the target nucleic acid sequence having the certain detection temperature is determined by a difference between the signal detected at one or more detection temperatures higher than the certain detection temperature and the signal detected at the certain detection temperature;
• wherein when the certain detection temperature is a relatively highest detection temperature among the detection temperatures, the presence of a target nucleic acid sequence is determined by the signal detected at the certain detection temperature.