Quadruple fluorescence quantitative kit for rapid detection of SNP (single nucleotide polymorphism) loci

A technology of fluorescence quantification and kit, applied in the direction of recombinant DNA technology, measurement/testing of microorganisms, DNA/RNA fragments, etc.

Active Publication Date: 2014-05-21
AGCU SCIENTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the principle of permutation and combination, SNP can have a total of 6 replacement situations, namely A / G, A / T, A / C, C / G, C / T and G / T, but in fact, the frequency of conversion is the majority, And it is mainly C / T conversion. The reason is that the C of CpG is methylated, and it is easy to spontaneously deaminate to form thymine T. Therefore, CpG becomes a mutation hotspot.

Method used

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  • Quadruple fluorescence quantitative kit for rapid detection of SNP (single nucleotide polymorphism) loci
  • Quadruple fluorescence quantitative kit for rapid detection of SNP (single nucleotide polymorphism) loci
  • Quadruple fluorescence quantitative kit for rapid detection of SNP (single nucleotide polymorphism) loci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Design of primers corresponding to each site in the kit, and determination of kit composition and amplification program

[0048] The design of primers and probes is directly related to the accurate typing of SNP loci. It complies with the design principles: ①The 5' end of the probe should be as close as possible to the upstream primer; ②Because of the fluorescence quenching effect of the G base, avoid using the G base at the 5' end; ③The C base of the entire probe is higher than the G base;④ Due to the specificity of using the probe to detect the SNP site, the probe contains the SNP site, and the SNP site is generally placed in front of the 2 bases at the 3' end of the probe ⑤The probe needs to be absolutely conservative. In addition to the distinction of the bases to be detected, the two probes for detecting the same SNP site are slightly different in length and constituent bases, so as to improve the uniqueness and specificity of the probes. ⑥The annealing ...

Embodiment 2

[0058] Example 2 SNP typing of human sample B in system 1

[0059] Reaction template extraction: use the magnetic bead method extraction kit to sample (FTA card, blood filter paper, etc.). It is advisable to add the lysate, cover the sample, and heat-treat at 95°C. After the cells are completely lysed, add magnetic beads to the lysate. Finally, place the magnetic beads with adsorbed DNA on the magnetic stand, discard the supernatant; add the lysate again, and repeat the previous steps; wash twice in detergent, and discard the supernatant. After the magnetic beads are dry, add DNA buffer and heat treatment at 56°C for 10 minutes to facilitate the dissolution of DNA. The supernatant obtained after centrifugation is the extracted DNA template.

[0060] For human sample B, the concentration is 0.5ng / μl, and the SNP typing results are detected. Take 2 μl to prepare the amplification system. The standard sample addition is also 2μl. The SNP typing sites rs921115 and rs1009480 ...

Embodiment 3

[0066] Embodiment 3 The effect of the present invention in forensic identification

[0067] One of the most important features of the kit provided by the invention is to assist STR detection results in forensic identification. Because the amplification is faster, the typing results can be obtained prior to STR capillary electrophoresis detection. Use the quadruple fluorescent quantitative kit for rapid detection of SNP sites that can simultaneously detect 10 SNP sites provided by the present invention to perform individual identification and identification on the criminal suspect who has been absconded for many years and the son of the suspect's hometown to determine the identity of the suspect. True identity (the corresponding amplification curve is shown in Figure 7-Figure 16 ). The composition and amplification procedure of the detection kit are the same as in Example 1, and the method in Example 2 is used to identify the relationship between the criminal suspect and the...

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Abstract

The invention provides a kit for detecting SNP (single nucleotide polymorphism) loci by directly using TaqMan probes, relating to ten autosomal SNP loci. Each SNP locus corresponds to two upstream and downstream primers and two specific TaqMan probes, ten SNP loci are divided into five sets of systems, and two SNP loci are detected simultaneously in a single-tube reaction system. The probes select four channels, namely, FAM, HEX, CY5 and ROX. The kit disclosed by the invention is implemented by adopting a quadruple probe detection technique, and through specific amplification, the information of two SNP loci is detected in a single reaction system. The kit is applied to forensic individual diagnosis, the detection is rapid, and the individual recognition rate is as high as 98.14%; because the kit is applicable to quantitative PCR (polymerase chain reaction) instruments of kits, the kit is easy to carry and can be directly applied to crime scenes for surveying.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a fluorescent quantitative PCR detection kit, in particular to a quadruple (four probes) real-time fluorescent quantitative PCR detection method for simultaneously detecting two SNP sites in the same reaction tube, detecting 10 sites in total SNP loci. It can be applied to forensic on-site rapid detection. Background technique [0002] SNP refers to a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, and the frequency of occurrence in the population is not less than 1%. Including single base transitions, transversions, insertions and deletions, etc. The so-called conversion refers to the conversion between the same type bases, such as the substitution between purine and purine (G→A), pyrimidine and pyrimidine (T→C); the so-called transversion refers to the substitution between purine and pyrimidine (A→T, A →C, C→G, G→T). According to the principl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2561/101C12Q2563/107
Inventor 郑卫国张帆郭育林卢青葛海鹏
Owner AGCU SCIENTECH
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