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Fluorescent quantitative kit for fast detecting SNP loci

A fluorescence quantitative and kit technology, applied in the field of molecular biology, can solve the problems of cross-contamination, low selectivity and low sensitivity of SNP site detection, and achieve the effects of short time, saving time and labor costs, and high sensitivity

Inactive Publication Date: 2019-06-21
湖南融健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, the present invention aims to propose a fluorescent quantitative kit for rapid detection of SNP sites to solve the problems of low selectivity, low sensitivity, and cross-contamination of SNP sites.

Method used

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  • Fluorescent quantitative kit for fast detecting SNP loci
  • Fluorescent quantitative kit for fast detecting SNP loci
  • Fluorescent quantitative kit for fast detecting SNP loci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Amplification and detection of gene Kras mutation site rs121913529

[0026] The mutant Kras plasmid DNA Mutant Target containing the rs121913529 site and the wild-type Kras plasmid DNA Wild Target containing the rs121913529 site were used as detection targets respectively to verify the sensitivity and specificity of the present invention. The Kras plasmid DNA sequence is shown in Table 1 (the DNA sequence shown and its complementary strand).

[0027] Table 1 Plasmid sequence

[0028]

[0029]

[0030] The primers in the kit are designed based on the Kras mutation site rs121913529, including the first type of primer DP1, the second type of primer P2, the third type of primers P3 and P4, wherein the rU in the first type of primer DP1 is a ribonucleotide, and Kras The target mutation site in the sequence is complementary, and C3Spacer is modified at the 3' end, which can block the extension reaction of the nucleic acid polymerase to the first primer DP1. ...

Embodiment 2

[0043] Example 2: Amplification and detection of the rs121913529 mutation site in cellular genomic DNA

[0044] 10 respectively 6 HT29 cells (rs121913529 mutation-negative) and SW480 cells (rs121913529 mutation-positive) were extracted with ONE-4-ALL Genomic DNA Mini-Preps Kit respectively, and then each took 1% or 0.1% of the cell extract, and carried out with the kit of the present invention For the amplification and detection of the rs121913529 mutation site, the amplification detection mixture used was the same as that in Example 1. For the real-time fluorescence curve of the analysis results, please refer to the attached Figure 4 . Real-time fluorescence curves showed that two different concentrations of rs121913529 mutation-positive cells SW480 gave real-time fluorescence curves with different POI values ​​in about 40 minutes, and different concentrations of rs121913529 mutation-negative cells HT29 did not give real-time fluorescence curves within 90 minutes. It show...

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Abstract

The invention provides a fluorescent quantitative kit for fast detecting SNP loci. The fluorescent quantitative kit comprises reaction mixed liquid and an operation instruction and is characterized inthat the reaction mixed liquid comprises first primers, second primers, third primers, a nucleotide monomer mixture, endoribonuclease, nucleic acid polymerase and nucleic acid double-strand fluorescent dye, the first primers are used for identifying the SNP loci, the second primers and the third primers are used for assisting the first primers to perform SNP characteristic sequence isothermal amplification, and the reaction system adopts a primer activated isothermal strand displacement nucleic acid amplification manner. The fluorescent quantitative kit for fast detecting the SNP loci has theadvantages that the kit is high in sensitivity, capable of detecting the target object of 22aM and good in selectivity and has mispairing distinguish rate of 10000 times, the reaction is completed inone step and is a closed-tube reaction, cross contamination is avoided, reaction time is short, fluorescent labeling is not needed, genome samples can be directly detected, and time and manpower costare saved.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a fluorescence quantitative kit for rapidly detecting SNP sites. Background technique [0002] Gene polymorphism widely exists in the genome of organisms, and single base polymorphism (SNP) is the most common form of gene polymorphism. SNP mainly refers to the DNA sequence polymorphism caused by a single nucleotide variation at the genome level. It is the most common type of human heritable variation, accounting for more than 90% of all known polymorphisms. SNP exists widely in the human genome and is a kind of gene mutation, mainly base substitution mutation, with an average of 1 in every 500-1000 base pairs, and the total number is estimated to be 3 million or more. SNP can provide important genetic data, which is extremely important for disease diagnosis and forensic analysis. [0003] At present, the existing SNP detection technologies at home and abroad mainly include the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
Inventor 蒋健晖王海波唐丽娟钟志坚李武
Owner 湖南融健生物科技有限公司
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