Detection technical system for enriching low-abundance DNA mutation based on nuclease-coupled PCR principle and application thereof

A nucleic acid and nucleic acid sample technology, applied in the biological field, can solve problems such as high equipment and reagent prices, high experimental operation requirements, and difficult synthesis of MGB probes

Active Publication Date: 2019-06-14
JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the synthesis of MGB probes is difficult and expensive, which is not conducive to wide application.
Digital PCR is another high-sensitivity detection technology that has emerged in recent years. This technology can reach a sensitivity of 0.01%, but this technology is prone to false positive results.
Similarly, high equipment and reagent prices and extremely high experimental operation requirements also limit its large-scale promotion

Method used

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  • Detection technical system for enriching low-abundance DNA mutation based on nuclease-coupled PCR principle and application thereof
  • Detection technical system for enriching low-abundance DNA mutation based on nuclease-coupled PCR principle and application thereof
  • Detection technical system for enriching low-abundance DNA mutation based on nuclease-coupled PCR principle and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Primer and detection probe design

[0107] Primer design follows the principles: primer requirements: ① avoid a series of bases in the primer sequence, especially a series of G; The last 5 bases at the 3′ end of the primer should not have more than 2 bases (G+C); ⑤The closer the downstream primer is to the probe, the better, and the fragments can overlap. The amplified fragment is preferably 75-150 bp.

[0108] The principle to be followed in the design of the detection probe: the detection probe specifically binds to the target gene, and its binding site is in any region of the target gene. The 5' end of the probe is labeled with a fluorescent reporter group (Reporter, R), such as FAM, VIC, etc., and the 3' end is labeled with a quencher group (Quencher, Q). Probe design requirements: ①The 5′ end of the probe cannot be G; ②The length of the probe should not be less than 13bp; ③Avoid a series of repeated base sequences; ④The Tm is 65-70°C, and the theoretical annealing...

Embodiment 2

[0112] Oligonucleotide gDNAs design and optimization

[0113] The core principle of this method for enriching low-abundance mutant target genes is that: on the one hand, the affinity of the PfAgo-gDNA complex to nucleic acid substrates is significantly improved by phosphorylation modification of the 5' end of gDNAs. At the same time, at the beginning of the establishment of this method, it was found that there is a seed region on the gDNA, and the specificity of the interaction between the PfAgo-gDNA complex and the substrate is determined by the seed sequence in gDNAs. This method explores the effect of different nucleotides (bases) on improving the specific targeting of the PfAgo-gDNA complex in conjunction with the target DNA substrate by exploring different positions (the second to the fifteenth nucleotides) in the gDNAs seed region and Its rules, analysis and induction of the design rules of the gDNAs seed region used to identify single nucleotide variation, are as follow...

Embodiment 3

[0120] Differential cleavage of ssDNA and dsDNA by PfAgo-gDNA complex

[0121] In this embodiment, it is mainly tested whether the PfAgo-gDNA complex still has a good ability to distinguish and cut ssDNA, dsDNA and dsDNA under the PCR working system under the common PCR reaction buffer and other components.

[0122] 3.1 Method

[0123]The components and working conditions involved in this example mainly include: 2×PCR Taq Master Mix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , templates (pure wild, pure mutant, and half of wild and mutant), etc., as shown in Table 8.

[0124] Table 8 takes the 25 μL system as an example, the components and preparation sequence of the PfAgo enrichment reaction system.

[0125] The description of each component in Table 8 is as follows:

[0126] 2×PCR Taq Master Mix reaction solution is prepared from 2×PCR buffer, dNTPs and hot start enzyme. 2×PCR buffer: KCl, (NH 4 ) 2 SO 4 , 3mM MgCl 2 , Tris-HCl, pH 8.3 (25...

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Abstract

The invention provides a detection system for enriching low-abundance mononucleotide mutant genes based on a nuclease-coupled PCR principle. The invention particularly provides a method for increasingthe relative abundance of a target nucleic acid, which comprises the following steps: (a) providing a nucleic acid sample containing a target nucleic acid and a non-target nucleic acid, wherein the abundance of the target nucleic acid in the nucleic acid sample is F1a; (b) using the nucleic acid in the nucleic acid sample as a template, performing PCR and nucleic acid cleavage reaction in the amplification-cleavage reaction system to obtain amplification-cleavage reaction product; the target nucleic acid is amplified the abundance of the amplification-cleavage reaction product is F1b, and theratio of F1b / F1a is more than or equal to 10. The detection system has the advantages of non-invasion, easy operation, high speed and the like, the sensitivity can reach 0.01%, and the DNA quantity of the sample can be reduced to aM grade.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection system for enriching low-abundance single nucleotide mutant genes (Single nucleotide variant, SNV) based on the principle of nuclease-coupled PCR. Background technique [0002] In recent years, the concept of "liquid biopsy" is emerging. The basic idea is to use blood and other body fluid samples instead of tumor tissue samples for pathology and molecular biology detection. Obtaining tumor gene mutation information has become a trend. Compared with the current standard tissue biopsy, the revolutionary liquid biopsy has the following irreplaceable advantages: less trauma, repeatability, homogenization heterogeneity, real-time judgment of efficacy, and dynamic adjustment of treatment decisions with the development of tumors. Therefore, the top ten breakthrough technologies of the year (Breakthrough Technologies 2015) released by MIT Technology Review in 2015, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q1/6844C12Q2521/301
Inventor 冯雁刘倩荀冠华郭翔李忠磊崇曰盛
Owner JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO LTD
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