| 摘要 |
The present invention discloses a method for detecting multiple single nucleotide variations or polymorphisms in a single reaction tube, and the oligonucleotide, the probe, the set of probes, the kit used, as well as the use thereof. Specifically, it relates to a method for identifying the genotype of multiple single nucleotide variation or SNP sites from the melting temperature of a kind of artificial melting temperature tag sequence (AMTS) and the type of fluorescence labels. |
| 主权项 |
1. A method for detecting single nucleotide polymorphisms (SNPs) at least at a first site and a second site in a template DNA in a single reaction tube, comprising:
(1) providing, in a single reaction tube:
(A) at least a first pair of hybridization probes and a second pair of hybridization probes, wherein:
(i) the first pair of hybridization probes comprises: (a) a first left hybridization probe which comprises, from its 5′ end to its 3′ end, a universal upstream primer binding sequence, a spacer sequence, a first artificial melting temperature tag sequence (AMTS) specific for the genotype of a first single nucleotide polymorphism (SNP) at the first site in the template DNA, and a first template hybridization sequence, wherein the base at the 3′ end of the first left hybridization probe is complementary to the nucleotide of the first SNP at the first site in the template DNA; and (b) a first right hybridization probe which comprises, from its 5′ end to its 3′ end, a second template binding sequence and a universal downstream primer binding sequence, wherein the first left hybridization probe and the first right hybridization probe are adjacent to each other on the template DNA after they hybridize to the template DNA and are capable of being ligated to each other on the template DNA by a DNA ligase; and(ii) the second pair of hybridization probes comprises: (a) a second left hybridization probe which comprises, from its 5′ end to its 3′ end, a universal upstream primer binding sequence, a spacer sequence, a second AMTS sequence specific for the genotype of a second SNP at the second site in the template DNA, and a third template hybridization sequence, wherein the base at the 3′ end of the second left hybridization probe is complementary to the nucleotide of the second SNP; and (b) a second right hybridization probe which comprises, from its 5′ end to its 3′ end, a fourth template binding sequence and the universal downstream primer binding sequence, wherein the second left hybridization probe and the second right hybridization probe are adjacent to each other on the template DNA after they hybridize to the template DNA and are capable of being ligated to each other on the template DNA by a DNA ligase, wherein the first site and the second site are located on different locations of the template DNA;(B) denatured template DNA;(C) a hybridization buffer;(D) a ligase; and(E) a ligation buffer; (2) performing hybridization and ligation reactions in the single reaction tube, thereby forming (a) a first complete single-stranded nucleic acid comprising the first left hybridization probe and the first right hybridization probe and (b) a second complete single-stranded nucleic acid comprising the second left hybridization probe and the second right hybridization probe; (3) performing a polymerase chain reaction (PCR) in the single reaction tube using a pair of universal primers, wherein the pair comprises (a) a universal upstream primer that hybridizes with the universal upstream primer sequence, and (b) a universal downstream primer that hybridizes with the universal downstream primer binding sequence, thereby producing the first complete single-stranded nucleic acid and the second complete single-stranded nucleic acid, wherein the first complete single-stranded nucleic acid comprises the first AMTS and the second complete single-stranded nucleic acid comprises the second AMTS; (4) forming a first duplex and a second duplex by either (i) performing the PCR in the presence of a fluorescent probe which is capable of hybridizing with the first AMTS and the second AMTS or (ii) adding the fluorescent probe into the single reaction tube after the PCR, wherein the first duplex comprises the first AMTS and the first fluorescent probe and wherein the second duplex comprises the second AMTS and the second fluorescent probe; and (5) generating fluorescent melting curves of the first duplex and the second duplex and performing melting curve analysis on the first duplex and the second duplex, thereby detecting the first SNP at the first site in the template DNA and the second SNP at the second site in the template DNA based on the heights of the melting curve peaks of the fluorescent melting curves of the first duplex and the second duplex. |
