Fusion primer combination capable of rapidly constructing amplicon library

An amplicon library and primer combination technology is applied in the field of fusion primer combinations for rapidly constructing amplicon libraries, and can solve the problems of high cost, easy contamination of the library, and high loss rate of the library

Inactive Publication Date: 2017-06-30
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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  • Fusion primer combination capable of rapidly constructing amplicon library
  • Fusion primer combination capable of rapidly constructing amplicon library
  • Fusion primer combination capable of rapidly constructing amplicon library

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Embodiment 1

[0098] 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:

[0099] 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 DNA extraction quality, after detecting the sample DNA concentration with Qubit3.0, dilute each genomic DNA sample to a concentration of 20ng / μl.

[0100] 2. The sequence information of the fusion primers used to construct the amplic...

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Abstract

The invention discloses a fusion primer combination capable of rapidly constructing amplicon library. The fusion primer combination is capable of fusing a primer according to an upstream fusion primer designed by target amplicon and a downstream fusion primer designed by target amplicon, wherein the upstream fusion primer designed by target amplicon comprises a first linking sequence, a barcode sequence, a second linking sequence and the specific upstream primer sequence designed by target amplicon arranged according to 5' to 3' direction in order; the downstream fusion primer designed by target amplicon comprises a third linking sequence and the specific downstream primer sequence designed by target amplicon arranged according to 5' to 3' direction in order; the fusion primer combination can subjected to one-step PCR construction to obtain the amplicon library in a simple and rapid mode, barcode is introduced when PCR is initiated, possibility of intersect pollution between a sample and the library are greatly reduced; partition requirement of the experiment places can be simplified, and the cost of library establishment of a single sample can be controlled in 30 yuan/example by the fusion primer combination.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a combination of fusion primers 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 traditi...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B50/06
CPCC12N15/1093C12N15/11C12Q1/6806C12Q1/6869C40B50/06C12Q2531/113C12Q2525/191C12Q2563/185
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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