Detection of target nucleic acid sequences using different detection temperatures and reference values

A method for SNP (single nucleotide polymorphism) genotyping of a nucleic acid sequence in a sample using different detection temperatures and reference values, comprising:

• (a) providing (i) a first reference value for a homozygote composed of a first SNP allele, wherein said first reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a first signal-generating means;
• (ii) a second reference value for a homozygote composed of a second SNP allele wherein said second reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a second signal-generating means;
• and (iii) a third reference value for a heterozygote composed of the first SNP allele and the second SNP allele wherein said third reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by the first signal-generating means and the second signal-generating means;
• wherein the three reference values are different from each other;
• (b) incubating the sample with the first signal-generating means and the second signal-generating means for the SNP alleles and detecting signals from the two signal-generating means at the relatively high detection temperature and the relatively low detection temperature;
• wherein the two signal-generating means generate signals at the relatively high detection temperature and at the relatively low detection temperature;
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• and
• (c) determining a SNP genotype by the reference values and a difference between the signals detected at the relatively high detection temperature and the relatively low detection temperature in the step (b).

A computer readable storage medium containing instructions to configure a processor to perform a method for determining the presence of at least one target nucleic acid sequence of two target nucleic acid sequences comprising a first target nucleic acid sequence and a second target nucleic acid sequence in a sample using different detection temperatures and reference values, the method comprising:

• (a) receiving signals in the sample generated from a first signal-generating means for the first target nucleic acid sequence and a second signal-generating means for the second target nucleic acid sequence at a relatively high detection temperature and a relatively low detection temperature;
• wherein the two signal-generating means generate signals at the relatively high detection temperature and the relatively low detection temperature;
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• and
• (b) determining the presence of at least one target nucleic acid sequence by the signals received in the step (a), and a first reference value for the first target nucleic acid sequence and/or a second reference value for the second target nucleic acid sequence;
• wherein the first reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a first signal-generating means, and the second reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a second signal-generating means;
• wherein the first reference value is different from the second reference value.

A computer readable storage medium containing instructions to configure a processor to perform a method for SNP (single nucleotide polymorphism) genotyping of a nucleic acid sequence in a sample using different detection temperatures and reference values, the method comprising:

• (a) receiving signals in the sample generated from a first signal-generating means and a second signal-generating means for SNP alleles at a relatively high detection temperature and a relatively low detection temperature;
• wherein the two signal-generating means generate signals at the relatively high detection temperature and at the relatively low detection temperature;
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• and
• (b) determining a SNP genotype by a difference between the signals received in the step (a), and a first reference value for a homozygote composed of a first SNP allele, a second reference value for a homozygote composed of a second SNP allele and a third reference value for a heterozygote composed of the first SNP allele and the second SNP allele;
• wherein the first reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a first signal-generating means, the second reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a second signal-generating means, and the third reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by the first signal-generating means and the second signal-generating means;
• wherein the three reference values are different from each other.

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 at least one target nucleic acid sequence of two target nucleic acid sequences comprising a first target nucleic acid sequence and a second target nucleic acid sequence in a sample using different detection temperatures and reference values, the method comprising:

• (a) receiving signals in the sample generated from a first signal-generating means for the first target nucleic acid sequence and a second signal-generating means for the second target nucleic acid sequence at a relatively high detection temperature and a relatively low detection temperature;
• wherein the two signal-generating means generate signals at the relatively high detection temperature and the relatively low detection temperature;
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• and
• (b) determining the presence of at least one target nucleic acid sequence by the signals received in the step (a), and a first reference value for the first target nucleic acid sequence and/or a second reference value for the second target nucleic acid sequence;
• wherein the first reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a first signal-generating means, and the second reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a second signal-generating means;
• wherein the first reference value is different from the second reference value.

A computer program to be stored on a computer readable storage medium to configure a processor to perform a method for SNP (single nucleotide polymorphism) genotyping of a nucleic acid sequence in a sample using different detection temperatures and reference values, the method comprising:

• (a) receiving signals in the sample generated from a first signal-generating means and a second signal-generating means for SNP alleles at a relatively high detection temperature and a relatively low detection temperature;
• wherein the two signal-generating means generate signals at the relatively high detection temperature and at the relatively low detection temperature:
• wherein signals to be generated by the two signal-generating means are not differentiated by a single type of detector;
• and
• (b) determining a SNP genotype by a difference between the signals received in the step (a), and a first reference value for a homozygote composed of a first SNP allele, a second reference value for a homozygote composed of a second SNP allele and a third reference value for a heterozygote composed of the first SNP allele and the second SNP allele;
• wherein the first reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a first signal-generating means, the second reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by a second signal-generating means, and the third reference value represents a relationship of change in signals provided at a relatively high detection temperature and a relatively low detection temperature by the first signal-generating means and the second signal-generating means;
• wherein the three reference values are different from each other.