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Low-frequency mutation detection method based on snapback primer technology

A technology of snapback probe primers and detection methods, which is applied in the field of gene detection, can solve problems such as obstacles, and achieve the effects of convenient operation, less pollution, and good enrichment and amplification

Inactive Publication Date: 2014-03-05
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ARMS technology requires clustering comparison, and has high requirements for the concentration and freshness of the original amplified genomic template DNA. For the detection of EGFR / KRAS gene mutations, the kit based on Scorpion-ARMS technology -- ADx-Kras kit It is currently recognized as the most sensitive method for EGFR / KRAS gene mutation, and the sensitivity for mutation detection can reach 1%, but this kit needs to design specific probes for different mutation detection
The results are greatly affected by the stability of the PCR instrument, the standard for judging the results of gene mutations, and the interpretation of the fluorescence curve. The technology uses clustering comparison to interpret the CT value, so the original template concentration needs to be homogenized, while serum and plasma samples The characteristics of this method make it an obstacle to apply this method to the detection of plasma and serum samples

Method used

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  • Low-frequency mutation detection method based on snapback primer technology
  • Low-frequency mutation detection method based on snapback primer technology
  • Low-frequency mutation detection method based on snapback primer technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Use the EGFR gene exon 19 deletion mutation sample, mix it with normal human sample DNA according to different ratios, and make the mutation accounted for 1 / 106, 1 / 105, 1 / 104, 1 / 1000, 1 / 100, 1 / 10 The mixed mutant samples, pure, mutant, and wild-type samples are amplified using the rebound probe common amplification method, and the enrichment amplification method is used for amplification, respectively, HRM detection is performed, and the enriched amplified PCR products are sent to sequencing. Detection sensitivity, enrichment efficiency.

[0061] 1.1. Preparation of DNA template:

[0062] 1.1.1 Use QIAamp DNA Mini Kit (Qiagen, Germany) to extract DNA from adjacent tissues and tumor tissues of lung cancer patients, use Nano Drop 2000 spectrophotometer to detect the concentration, and dilute to 20ng / μl with sterile double-distilled water, and obtain by sanger sequencing Wild-type sample, mutant sample (15bp deletion).

[0063] 1.1.2 Mix and match the same concentration (20ng / μ...

Embodiment 2

[0081] Use the EGFR gene L858R site mutation sample, mix it with normal human sample DNA according to different proportions, and make the mutation account for 1 / 10 6 , 1 / 10 5 , 1 / 10 4 , 1 / 1000, 1 / 100, 1 / 10 ratios of mutant mixed samples, pure, mutant, and wild-type samples are amplified using the rebound probe common amplification method, and the enrichment amplification method is used for HRM detection. , And send the enriched and amplified PCR products for sequencing. Detection sensitivity, enrichment efficiency.

[0082] 2.1. Preparation of DNA template:

[0083] 2.1.1 Use QIAamp DNA Mini Kit (Qiagen, Germany) to extract DNA from adjacent tissues and tumor tissues of lung cancer patients, use Nano Drop 2000 spectrophotometer to detect the concentration, and dilute to 20ng / μl with sterile double-distilled water, and sequence by sanger Obtain wild-type samples, mutant samples (L858R).

[0084] 2.1.2 Mix equal concentrations (20ng / μl) of mutant and wild-type DNA according to the re...

Embodiment 3

[0104] Use KRAS gene locus mutation samples, mix with normal human sample DNA in different proportions, and make mutations account for 1 / 10 6 , 1 / 10 5 , 1 / 10 4 , 1 / 1000, 1 / 100, 1 / 10 ratios of mutant mixed samples, pure, mutant, and wild-type samples are amplified using the rebound probe common amplification method, and the enrichment amplification method is used for HRM detection. , And send the enriched and amplified PCR products for sequencing. Detection sensitivity, enrichment efficiency.

[0105] 3.1. Preparation of DNA template:

[0106] 3.1.1 Use QIAamp DNA Mini Kit (Qiagen, Germany) to extract DNA from adjacent tissues and tumor tissues of lung cancer patients, use Nano Drop 2000 spectrophotometer to detect the concentration, and dilute to 20ng / μl with sterile double-distilled water, and obtain by sanger sequencing Wild type sample, mutant sample (G12A).

[0107] 3.1.2 Mix the mutant and wild-type DNA with equal concentration (20ng / μl) according to the required ratio to obta...

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Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a low-frequency mutation detection method based on a snapback primer technology. The method comprises: designing a snapback primer, preparing a DNA template, preparing a detection system, setting a reaction procedure and determining a Ts value, judging a result, and other steps. According to the present invention, the detection method is the snapback ARMS and snapback primer HRM combined low-frequency mutation detection technology, such that the detection sensitivity of 1:10000 or even higher is achieved, clinical requirements of pollution resistance, high sensitivity, strong specificity, easy performing and result judgment on the mutation detection technology are met, and the method can be used for gene screening, genotyping (SNP, deletion mutation, insertion mutation) and other fields of various types of tissues including fresh tissue samples, whole blood, serum, tissue slices and other samples.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a low-frequency mutation detection method. Background technique [0002] With the increasing use of genetic testing technology in clinical practice, genetic testing, especially genotyping technology has been developed rapidly, providing a great opportunity for the diagnosis of genetic diseases, the detection of tumor marker genes, and the personalized medicine of tumor patients. available testing options. In the process of tumor-related gene detection, it was found that due to the heterogeneity of tumor tissue and the uncertainty of sampling positions, the sensitivity of tumor tissue detection technology is relatively high. [0003] In addition, many studies have found that circulating DNA is consistent with tissue DNA mutations, which indicates that the detection of peripheral free DNA in tumor patients can reflect the mutation status of tumor tissues, making...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2527/107C12Q2531/107C12Q2525/117
Inventor 孙海燕卢大儒
Owner FUDAN UNIV
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