Method for quickly building amplicon library

An amplicon library and amplicon technology are applied in the field of rapid construction of amplicon libraries, which can solve the problems of high cost, high library loss rate, and easy contamination of the library.

Inactive Publication Date: 2017-08-04
GENETRON HEALTHBEIJING LAB CO LTD
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Problems solved by technology

However, the traditional method of constructing an amplicon library on the LifeNGS platform is cumbersome and requires about 5 hours of PCR amplification, digestion, adapter addition, and purification; and because the multi-step operation needs to be uncapped, the library is easily destroyed. Contamination, high library loss rate; In addition, in the traditional method of constructing amplicon library, the cost of building a library for a single sample is relatively high, ranging from 200-1000 yuan / case

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  • Method for quickly building amplicon library
  • Method for quickly building amplicon library
  • Method for quickly building amplicon library

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Experimental program
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Embodiment 1

[0105] The samples to be tested are 5 FFPE samples (ie: formalin-fixed paraffin-embedded samples, FFPE stands for Formalin-Fixed and Parrffin-Embedded), of which 3 are FFPE samples from patients with non-small cell lung cancer, and the other 2 are non-small cell lung cancer samples. FFPE samples from tumor patients. Using specifically designed fusion primers, a one-step method was used to construct an amplicon DNA library for 5 FFPE samples. The specific process is as follows:

[0106] 1. Genomic DNA extraction: Use the Qiagen FFPE DNA Kit (spin column type) kit to extract the genomic DNA in the FFPE sample. For specific steps, refer to the kit operation instructions, dissolve the obtained genomic DNA in Tris-HCl buffer, and detect with Nano Drop For the quality of DNA extraction, after detecting the DNA concentration of the sample with Qubit 3.0, each genomic DNA sample was diluted to a concentration of 20ng / μl.

[0107] 2. The sequence information of the fusion primers used...

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Abstract

The invention discloses a method for quickly building an amplicon library. The method comprises the following steps of (1) synthesizing a fusion primer combination for building the amplicon library of a DNAA sample; (2) building a PCR reaction system of the DNA sample, mixing an upstream fusion primer designed according to a target amplicon and a downstream fusion primer designed according to the target amplicon as a primer combination in the PCR reaction system; and (3) carrying out PCR. According to the method, the amplicon library can be simply and quickly built through PCR in the step (1); a barcode is introduced at the beginning of the PCR, so that the possibility of cross contamination between samples and the library is greatly reduced and the requirements of experiment site partition can be simplified. According to the method, the cost of building the library for a single sample can be controlled within 30 yuan / case.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for rapidly constructing an amplicon library. Background technique [0002] Due to the characteristics of high throughput, high sensitivity, and high automation, next-generation sequencing (NGS) has been increasingly used in the research, diagnosis and treatment of diseases in recent years. NGS technology can realize multi-gene parallel detection, save samples compared with traditional detection methods, and has higher sensitivity, and can more truly restore the panorama of tumor mutations. However, the traditional method of constructing an amplicon library on the LifeNGS platform is cumbersome and requires about 5 hours of PCR amplification, digestion, adapter addition, and purification; and because the multi-step operation needs to be uncapped, the library is easily destroyed. Contamination, high library loss rate; In addition, in the traditional method of construc...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2531/113C12Q2525/191C12Q2563/185
Inventor 阎海王思振焦宇辰徐大勇郑乔松师晓
Owner GENETRON HEALTHBEIJING LAB CO LTD
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