Composition for detecting single nucleotide polymorphism an application of composition

A single nucleotide polymorphism and composition technology, which is applied in the field of gene fluorescent probe detection, can solve the problems of time-consuming, time-consuming, labor-intensive, and difficult operations, and achieves a reduction in the probability of non-specific binding, design difficulty, and development savings. cost effect

Active Publication Date: 2020-11-27
浙江绍兴鼎晶生物医药科技股份有限公司
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Problems solved by technology

Among them, the most common method is the sequencing method, which is relatively low in cost, but takes a long time to operate and has low sensitivity. Due to the limitation of sequencing instruments, it is difficult to popularize; The method of sequence determination is expensive and complicated; the detection sensitivity is low; the repeatability is poor; the analysis range is narrow
The high-resolution melting curve method only needs common fluorescent dyes and does not require specific probes. However, since the SNP site is only a mutation of one base, the requirements for equipment are relatively special, and there are certain difficulties in clinical promotion.
The traditional fluorescent quantitative PCR method is widely used in clinical practice, but the probe design of this method is difficult and requires more optimization design work, which is time-consuming and labor-intensive. It is more difficult to design special templates such as hig

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  • Composition for detecting single nucleotide polymorphism an application of composition
  • Composition for detecting single nucleotide polymorphism an application of composition
  • Composition for detecting single nucleotide polymorphism an application of composition

Examples

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Example Embodiment

[0043] Example 1: as figure 1 As shown, the allele-specific primers and their associated probes were designed and used for the detection of the ApoE gene rs429358 (T / C) SNP locus; the PCR template was cell line genomic DNA.

[0044] Reaction liquid system preparation: H 2 O, purified water, 16.7 μL; PCR buffer (Mg 2+ plus), 5X, 5 μL; dNTPs (each 10 mM dATP, dCTP, dGTP; 20 mM dUTP), 10 mM, 0.5 μL; allele-specific primer 1, 100 uM, 0.1 μL; allele-specific primer 2, 100 uM, 0.1 μL; Allele-specific primer-associated probe 1, 100uM, 0.05μL; allele-specific primer-associated probe 2, 100uM, 0.05μL; upstream primer, 100uM, 0.1μL; downstream primer, 100uM, 0.1μL; UDG enzyme, 1U / μL, 0.1 μL; polymerase, 5U / μL, 0.2 μL; total volume 23 μL.

[0045] Operation steps: Prepare the relevant liquids according to the above reaction solution system, shake and mix for 15 seconds, centrifuge quickly for 15 seconds, dispense 23 μL per tube into PCR reaction tubes, add 2 μL of the samples to be ...

Example Embodiment

[0059] Example 2: as figure 2 As shown, allele-specific primer-complementary probe PCR detection technology was used for the detection of MTHFR gene 677 (C / T) SNP locus; the PCR template was cell line genomic DNA.

[0060] Reaction liquid system preparation: H 2 O, purified water, 16.7 μL; PCR buffer (Mg 2+ plus), 5X, 5 μL; dNTPs (each 10 mM dATP, dCTP, dGTP; 20 mM dUTP), 10 mM, 0.5 μL; allele-specific primer 1, 100 uM, 0.1 μL; allele-specific primer 2, 100 uM, 0.1 μL; Allele-specific primer-associated probe 1, 100uM, 0.05μL; allele-specific primer-associated probe 2, 100uM, 0.05μL; upstream primer, 100uM, 0.1μL; downstream primer, 100uM, 0.1μL; UDG, 1U / μL, 0.1 μL; polymerase, 5U / μL, 0.2 μL; total volume 23 μL.

[0061] Operation steps: Prepare the relevant liquids according to the above reaction solution system, shake and mix for 15 seconds, centrifuge quickly for 15 seconds, dispense 23 μL per tube into PCR reaction tubes, add 2 μL of the samples to be tested, and then...

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Abstract

The invention belongs to the technical field of fluorescent probe detection of genes, and particularly relates to a composition for detecting single nucleotide polymorphism and an application of the composition. The composition for detecting single nucleotide polymorphism comprises an upstream primer and a downstream primer which are shared among different alleles, allele specific primers for eachspecific allele, and probes being respectively complementary and associated with various allele specific primers to use different fluorescence labelling, wherein the probes cover single nucleotide polymorphism sites. The composition for detecting single nucleotide polymorphism is adopted for gene detection, so that single-pipe multi- allele detection can be realized, the sensitivity is high, heterozygosis or homozygosis types of the genes can also be distinguished, repeated optimization of the probe is not needed, the cost is saved, the detection flux can be increased, the results are accurate and analysis is facilitated.

Description

technical field [0001] The invention belongs to the technical field of gene fluorescent probe detection, and in particular relates to a single nucleotide polymorphism detection composition and application thereof. Background technique [0002] Real-time fluorescent quantitative PCR technology was introduced by Applied Biosystems of the United States in 1996. It is a specific fluorescent probe added while adding a pair of primers during PCR amplification. The probe is an oligonucleotide. A reporter fluorophore and a quencher fluorophore are labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme digests and hydrolyzes the probe, so that the reporter fluorescent group and the quencher group The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DN...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/125C12Q2563/107C12Q2547/101
Inventor 焦海涛何志辉葛海鹏沈伟强
Owner 浙江绍兴鼎晶生物医药科技股份有限公司
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