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Next generation sequencing primer probe set and detection method of microsatellite instability state

A microsatellite instability, next-generation sequencing technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of complex operation of immunohistochemical methods, long period of direct sequencing, and low specificity, etc. Achieve the effect of reducing the cost of library construction, low reagent requirements, and simplifying detection steps

Pending Publication Date: 2018-12-25
上海赛安生物医药科技股份有限公司
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Problems solved by technology

Immunohistochemical method is complicated to operate and has low specificity; microsatellite site PCR method has low throughput; direct sequencing method has long period, high cost and low accuracy

Method used

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  • Next generation sequencing primer probe set and detection method of microsatellite instability state
  • Next generation sequencing primer probe set and detection method of microsatellite instability state
  • Next generation sequencing primer probe set and detection method of microsatellite instability state

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Embodiment

[0041] For the second-generation sequencing detection method of microsatellite instability state in this embodiment, the detection sample can be any form of sample that can extract nucleic acid, including but not limited to: whole blood, serum, plasma and tissue samples; tissue samples include but not Limited to: paraffin-embedded tissue, fresh tissue, and frozen sections.

[0042] 1. Sample extraction.

[0043] A kit (AxyPrep Multisource Genomic DNA Miniprep Kit) was used to extract tumor tissue slices and paracancerous tissue slices from a patient with colorectal cancer. For specific operations, refer to the kit product manual.

[0044] 2. Determination of sample DNA concentration.

[0045] use 3.0 For the determination of the DNA concentration after extraction, the specific steps are as follows:

[0046] 1) Add 1ul Qubit dsDNA HS Reagent and 199ul Qubit dsDNA HSbuffer to the sample tube, vortex and mix for 4s;

[0047] 2) Discard 1 μl of the working solution into the s...

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Abstract

The invention relates to a next generation sequencing detection method of a microsatellite instability state. The method includes: performing DNA fragmentation on the cancer tissue sample and normal tissue reference substance of a tumor patient, polishing and adding tail A to tail ends, and performing joint connection; designing primers and probes for target area capturing and primers for amplification, the probes are one-way DNA extension probes aiming at five microsatellite loci, and using the probes and the primers to perform target area capturing and then performing amplification to obtaina library; sequencing to obtain the repeated sequence length distribution data of the five microsatellite loci of the cancer tissue sample and the normal tissue reference substance, and comparing thedata of the five microsatellite loci of the cancer tissue sample and the normal tissue reference substance to judge the stability of the microsatellite loci. The invention further relates to a next generation sequencing primer probe set of the microsatellite instability state. The next generation sequencing detection method can reduce influence of sequencing depth on result judgement and is simple, practicable, high in accuracy, high in sensitivity and low in cost.

Description

technical field [0001] The invention relates to a second-generation sequencing method for evaluating microsatellite instability level and primers and probes used therefor, belonging to the field of biotechnology. Background technique [0002] Microsatellites refer to short repetitive DNA sequences on the genome, including mononucleotide repeats, dinucleotide repeats and polynucleotide repeats. These repeat regions are prone to cause base pair mismatches during DNA replication, resulting in changes in the length of the repeat regions. However, there is a robust mismatch repair (MMR) system in normal humans to repair the consequences of mismatches. [0003] Microsatellite instability (MSI) refers to changes in the length of DNA repeat regions that result from the loss of function of MMR proteins and occurs in up to 20% of colorectal cancer patients. MSI can be further divided into high instability (MSI-H) and low instability (MSI-L). More than or equal to 30% of the detected...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869
CPCC12Q1/6886C12Q1/6869C12Q2525/191
Inventor 赵新泰王明潘文健
Owner 上海赛安生物医药科技股份有限公司
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