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Tumor circulating DNA KRAS mutation detection method

A technology for tumor patients and tumor cells, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of insufficient sensitivity

Inactive Publication Date: 2015-07-08
SHANGHAI IND TECH INST +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The invention discloses a detection method for detecting KRAS second exon gene mutation through second-generation high-throughput sequencing of tumor circulating DNA, so as to overcome the shortcomings of insufficient sensitivity of traditional sequencing methods

Method used

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  • Tumor circulating DNA KRAS mutation detection method
  • Tumor circulating DNA KRAS mutation detection method
  • Tumor circulating DNA KRAS mutation detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The SEQ ID NO:1-4 of the present invention was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0052] The second exon of KRAS in 18 colorectal cancer tissues and paired circulating DNA was sequenced on the ABI 3730 sequencer. We collected frozen tissues of 18 cases of colorectal cancer operated in 2014 and extracted tissue DNA; extracted peripheral blood from patients, separated plasma, and extracted circulating DNA from tumors. The detection product of SEQ ID NO: 5 was obtained by primers 1 and 2 (PCR reaction conditions: 95°C for 15min; 94°C for 30s, 62°C for 30s, 72°C for 30s, 12 rounds, each round -0.5°C; 94°C for 30s, 56°C for 30s, 72°C for 30s, 28 rounds; 72°C for 7min, 4°C∞). The PCR products were purified, sequenced on the ABI 3730 sequencer, and the sequencing results were compared.

[0053] We found mutations in 8 samples in tumor tissue and 3 mutations in circulating DNA. The overall consistency rate of the two reached 83.3%, the positive sample consistency rate was 37.5%, and the correlation coefficient was 0.5 (Table 1).

[0054] Table 1: Comparison of t...

Embodiment 3

[0058] Using the same sample as in Example 2, obtain SEQ ID NO: 5 through primers 3 and 4, detect the product by electrophoresis, use AMPure XP beads (magnetic beads) to purify, and use PicoGreen fluorescent dye for accurate DNA quantification; add index linker: Index Kit-PCR primers (FC-121-1012, FC-121-1011), perform a second PCR reaction, and then use AMPure XP beads (magnetic beads) to purify again, use PicoGreen fluorescent dye for accurate DNA quantification, dilute to 0.015nM, 1ul sample loading. The MiSeq sequencer performs high-depth sequencing of PCR products; and performs data results.

[0059] We found that there were 8 cases of mutations in tumor tissue and 7 cases of mutations in circulating DNA, the overall agreement rate of the two was 94.4%, the agreement rate of positive samples was 87.5%, and the correlation coefficient was 0.892 (Table 2).

[0060] Table 2: Comparison of tumor tissue and circulating DNA results based on next-generation sequencing

[0061...

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Abstract

The invention relates to a tumor circulating DNA KRAS mutation detection method. The invention specifically relates to primer sequences (SEQ ID NO: 3 and 4) used for the detection, a kit and compositions comprising the primer sequences, an application of the primer sequences, and the like. According to the invention, with a detection method for detecting KRAS second exon gene mutation through second-generation high-throughput sequencing aiming at the tumor circulating DNA, defects such as inadequate sensitivity of traditional sequencing methods can be overcome.

Description

technical field [0001] The invention relates to a method for detecting KRAS mutation in tumor circulating DNA. Background technique [0002] Mutation detection based on high-throughput sequencing of peripheral blood circulating DNA has strong specificity and high sensitivity. Through the mutation detection of peripheral blood circulating DNA KRAS, the impact of overtreatment and its potential side effects can be avoided, while other patients can be treated more aggressively. Further, a mathematical model of dynamic evolution can be constructed to determine the best personalized cancer treatment plan. [0003] K-ras is a proto-oncogene, about 35kb in length, located on chromosome 12. It is a member of the RAS gene family and encodes K-ras protein, which is related to tumor formation, proliferation, migration, spread and angiogenesis. K-ras gene (K-ras, p21) family has three genes related to human tumors——H-ras, K-ras and N-ras, which are located on chromosome 11, 12 and 1, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 王志敏徐烨黄薇蔡三军李聪
Owner SHANGHAI IND TECH INST
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