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Primer combination for rapidly constructing amplicon library through one-step process

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

Active Publication Date: 2017-06-13
GENETRON HEALTH (BEIJING) CO LTD
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  • Claims
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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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  • Primer combination for rapidly constructing amplicon library through one-step process
  • Primer combination for rapidly constructing amplicon library through one-step process
  • Primer combination for rapidly constructing amplicon library through one-step process

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Embodiment 1

[0104] The samples to be tested are 6 FFPE samples (namely: formalin-fixed paraffin-embedded samples, FFPE stands for Formalin-Fixed and Parrffin-Embedded), of which 4 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 6 FFPE samples. The specific process is as follows:

[0105]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.

[0106] 2. Design and synthesize primers:

[0107] An upst...

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Abstract

The invention discloses a primer combination for rapidly constructing an amplicon library through a one-step process. The primer combination comprises a forward fusion primer designed according to a target amplicon, a reverse fusion primer designed according to the target amplicon, a forward universal primer and a reverse universal primer, wherein the forward fusion primer comprises a first linker sequence and a specific forward primer sequence designed according to the target amplicon; the reverse fusion primer comprises a second linker sequence and a specific reverse primer sequence designed according to the target amplicon; the forward universal primer comprises a third linker sequence, a barcode sequence and the first linker sequence; and the reverse universal primer comprises a universal sequence and the second linker sequence. According to the fusion primer combination, the amplicon library can be simply and rapidly constructed through one step, and before the PCR is started, the barcode is introduced, so the possibility of cross contamination between the samples and the library is greatly reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer combination for rapidly constructing an amplicon library in one step. 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 m...

Claims

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Application Information

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IPC IPC(8): C12N15/11C40B50/06
CPCC12N15/1093C12Q1/6869C40B50/06C12Q2535/122C12Q2525/191
Inventor 阎海王思振焦宇辰徐大勇郑乔松师晓
Owner GENETRON HEALTH (BEIJING) CO LTD
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