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Method for detecting mutation of multiple gene loci by using high-throughput sequencing technology

A technology for technical detection and site mutation, which is applied in the field of multi-gene site mutation detection using high-throughput sequencing technology, which can solve problems such as difficulty in detecting low-frequency or rare mutations, systematic errors, etc., so as to reduce detection costs and errors. rate, the effect of ensuring accuracy

Pending Publication Date: 2022-07-22
上海君远生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at the same time, it cannot be ignored that the systematic error introduced by the NGS platform makes it difficult for us to detect low-frequency or rare variants

Method used

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  • Method for detecting mutation of multiple gene loci by using high-throughput sequencing technology
  • Method for detecting mutation of multiple gene loci by using high-throughput sequencing technology
  • Method for detecting mutation of multiple gene loci by using high-throughput sequencing technology

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Experimental program
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Embodiment 1

[0035] see figure 1 , this implementation discloses a specific probe structure, and the probe is sequentially Blocker and Reporter parts from the 5' end to the 3' end. The Tm value of the specific probe was 15-25°C higher than that of the universal primer at the I5 end.

[0036] The specific probe can be modified by any one of thio-modification, deoxyuracil modification, deoxyhypoxanthine modification, 2-methoxy modification and phosphorylation modification, or it can be modified in multiple ways at the same time.

[0037] Wherein, the length of the Blocker part is 15-20 bases, including artificially introduced 2-6 arbitrary base sequences, other sites are complementary to the wild-type DNA sequence to be tested, and the 5' end of the Blocker part introduces a stem-loop structure, And the length of the stem-loop structure is 3-10 bases, and its sequence is complementary to a part of the Blocker sequence; the Reporter part is 20-30 bases in length, and its sequence is compleme...

Embodiment 2

[0052] This embodiment specifically discloses a specific detection method by taking the detection of DNA mutation in peripheral blood circulating tumor cell samples as an example

[0053] 1) End repair

[0054] This part is divided into two steps. First, end-repair of cfDNA is performed under the action of DNA polymerase to make double-stranded DNA form blunt ends, and then phosphorylation at the 5' end and addition of dA tail at the 3' end are performed respectively.

[0055] Prepare the reaction system in the following table:

[0056] component volume cfDNA XμL Nuclease-free water (40-X)μL Fragment and End Repair Buffers 10μL Fragments and end repair enzymes 10μL total capacity 60μL

[0057] After the preparation is completed, mix well and centrifuge, and use a common PCR machine. The reaction procedure is shown in the following table:

[0058] temperature time 20℃ 30 minutes 65℃ 30 minutes 4℃ ...

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Abstract

The invention relates to a method for detecting multi-gene locus mutation by using a high-throughput sequencing technology, which integrates the respective advantages of two library building modes of amplicon and hybrid capture, does not need the design of a capture probe and the enrichment of a target sequence by streptavidin magnetic beads, and has the advantages of lower detection cost, shorter library building period and higher detection efficiency. Meanwhile, good specificity and sensitivity can be ensured; under an asymmetric amplification system, a specific probe is introduced, and amplification of a wild type sequence can be effectively inhibited, so that a very high sequencing depth can be obtained by only needing a very small amount of sequencing data, the detection cost is greatly saved, and meanwhile, detection of a low-abundance mutant sequence is more facilitated; meanwhile, two rounds of PCR reaction are used, so that the occurrence of a nonspecific amplification phenomenon is greatly reduced; besides, the invention also uses a detection method of anchoring multiple PCR combined with high-throughput sequencing, has good advantages in the aspect of fusion gene detection, can detect unknown gene fusion, and greatly extends the application scene.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing, and in particular relates to a method for detecting multi-gene site mutation by using high-throughput sequencing technology. Background technique [0002] Cancer is the number one killer that threatens human health and life safety. With the completion of the Human Genome Project and the arrival of the post-genome era, cancer treatment methods are not limited to surgery, radiotherapy, chemotherapy, traditional Chinese medicine and biological therapy. In recent years, The emerging targeted therapy has shown obvious advantages and occupies an important position. [0003] Targeted therapy is an important treatment method in comprehensive tumor therapy. It refers to the development of effective blockers by targeting the landmark molecules of tumor cells to interfere with all aspects of cell canceration. Targeted therapy is significantly different from traditional radiotherapy and chemotherapy...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6869
CPCC12Q1/6886C12Q1/6869C12Q2600/156C12Q2531/113C12Q2523/308C12Q2535/122C12Q2525/191
Inventor 周文刚崔帆
Owner 上海君远生物科技有限公司
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