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Method of target region multiplex PCR and rapid library construction

A library construction and library technology, applied in the fields of genetics and precision medicine, to achieve high specificity, improve data utilization, library quality, and high uniformity

Active Publication Date: 2018-06-01
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to quickly construct a DNA library through multiple PCR and solve the consistency problem of multiple PCR amplification efficiency therein

Method used

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  • Method of target region multiplex PCR and rapid library construction
  • Method of target region multiplex PCR and rapid library construction
  • Method of target region multiplex PCR and rapid library construction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 multiplex PCR amplification

[0051] Step 1: Obtain the DNA of the sample

[0052]Whole blood was used as the sample, and cfDNA was extracted with an appropriate commercially available conventional cfDNA extraction kit, and extracted according to the instructions.

[0053] Step 2: The first round of PCR enrichment of target fragments

[0054] Prepare PCR reaction buffer system according to the table below

[0055]

[0056] Note: X and Y are adjusted according to the primer mix stock concentration (usually 100uM) and the number of primer pairs

[0057] The forward primer mix and reverse primer mix used in this example include a total of 92 pairs of specificity designed for human lung cancer mutation hotspots (such as EGFR gene p.E746_A750delELREA, p.V769_D770insASV, p.T790M, p.C797S, p.L858R) Primers, the specific primers include 5' modified anchor linker sequence + specific sequence for the region to be amplified, the 5' modified anchor linker sequenc...

Embodiment 2

[0077] Example 2 library construction and detection

[0078] Step 1: Purification

[0079] 1) Transfer the amplified product to a centrifuge tube, take 0.8× (40uL) Ampure XP magnetic beads or CMpure magnetic beads, mix with the amplified product and place it on a magnetic stand for 10 minutes;

[0080] 2) After the magnetic beads are completely adsorbed to the tube wall (about 5 min), discard the supernatant, wash the magnetic beads twice with freshly prepared 80% ethanol, and discard the supernatant;

[0081] 3) Let stand at room temperature for 5 minutes, and after the magnetic beads are dry (please be careful not to crack the magnetic beads due to excessive drying, so as not to affect the recovery efficiency), resuspend the magnetic beads with 17.5uL TE buffer, EB buffer or nucleic acid-free water according to downstream needs;

[0082] 4) After standing at room temperature for 5 minutes, place the centrifuge tube on a magnetic stand, and absorb 15uL of supernatant, which ...

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Abstract

The invention belongs to the field of genetics and precision medicines and relates to a novel method of targeted specific segment ultrahigh multiple PCR and rapid library construction. According to the method provided by the invention, a high quality library which meets the downstream analysis requirements can be acquired by taking 2.5-50ng extracted DNA as a template within 2-3h, and sample differentiation barcode is automatically added at a primer end in the library building process without secondary library building; amplicons of offload data are high in uniformity, hotspots on 99.5 percentof the amplicons reach 1200X coverage in the premise of 1M Reads; and the specificity is high, and 100 percent of a target sequence can all be effectively amplified; and addition of a library joint is directional, so that the data utilization ratio can be increased, and the library quality can be improved.

Description

technical field [0001] The invention belongs to the field of genetics and precision medicine, and relates to a new method for ultra-high multiplex PCR and rapid library construction targeting a specific segment. Background technique [0002] The greatest value of multiplex PCR technology in the field of genetics and precision medicine lies in its ability to improve the sensitivity of diagnosis. Sensitivity can be analyzed from two aspects: detection sensitivity and clinical sensitivity. [0003] Detection sensitivity emphasizes the detection result: if the sample contains the detected substance, what is the probability of the detection being successful. In the case of a PCR reaction, this is the lowest copy number that can be detected. Clinical sensitivity is to look at the problem from a clinical point of view: how high is the success rate of the experimental results in diagnosing the patient. If a disease has only one etiology, there is no difference in detection sensit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/686C40B50/06
CPCC12Q1/6806C12Q1/686C40B50/06C12Q2525/191C12Q2537/143
Inventor 胡春旭陆思嘉任军
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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