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Multiple PCR method for improving amplification specificity and uniformity

A specific and multiple technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems that the specificity and uniformity of amplification cannot be guaranteed, and achieve the effect of improving amplification efficiency and quality

Active Publication Date: 2019-12-20
GENETALKS BIO TECH CHANGSHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to break through the above-mentioned difficulties of multiplex PCR, many studies have optimized and adjusted the key link of primer design, and achieved some breakthroughs. However, when the number of amplification primers increases to a certain number, the amplification specificity and uniformity cannot be guaranteed.

Method used

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  • Multiple PCR method for improving amplification specificity and uniformity
  • Multiple PCR method for improving amplification specificity and uniformity
  • Multiple PCR method for improving amplification specificity and uniformity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Primer Design

[0038]According to NCBI's SNP database, search for a total of 184 target segment sequences carrying SNP sites in the human genome related to tumor risk, health risk, genetic disease, nutritional metabolism, drug metabolism, etc., use Primer5 primer design software to design primers, and then The upstream and downstream specific primers (as shown in Table 1 below) were synthesized by chemical methods, and the primer synthesis was completed by Shanghai Sangon Bioengineering Co., Ltd.

[0039] Universal adapters are added to the 5' ends of each pair of specific upstream primers and downstream primers that have been screened or designed. The sequence of the universal adapter (Universal adapter-F) of the upstream primer is 5'-AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAG AGACAG-3' (SEQ ID NO: 1). The universal adapter (Universal adapter-R) of the downstream primer consists of three parts. Starting from the 5' end, it is the next-generat...

Embodiment 2

[0051] Embodiment 2. Preparation of template DNA

[0052] Extraction of WBC Genomes from 45 Persons to be Examined

[0053] Genome extraction kit (purchased from QIAOGEN) was used, and the genome of leukocytes was extracted according to the instruction manual of the kit, and the specific steps were as follows:

[0054] (1.1) Thaw the blood sample at room temperature, mix well, and spin off, take 100 μl of white blood cells to a 1.5ml centrifuge tube, add 100 μl of PBS, mix well, and spin off;

[0055] (1.2) Add 10 μl RNaseA, 20 μl proteinase K, vortex and mix, let stand for 1 min, add 200 μl BufferAL, mix and centrifuge;

[0056] (1.3) Metal bath 56°C, 10min;

[0057] (1.4) Take it off, after cooling down to room temperature, add 200 μl absolute ethanol, mix well, and spin off;

[0058] (1.5) Transfer to the spin column, place at room temperature for 5min, 10000g, 1min, discard the filtrate;

[0059] (1.6) Add 500μl Buffer AW1, 10000g×1min, discard the filtrate;

[0060] ...

Embodiment 3

[0077] Example 3. Multiplex PCR reaction

[0078] 45 blood samples were collected, and each sample was processed according to the above method. The index sequence corresponding to each sample is shown in Table 2.

[0079] Table 2 Sample number and its corresponding index sequence

[0080] sample number Index sequence sample number Index sequence sample number Index sequence SGC01 CCTTAAT SGC16 GCATTGG SGC31 GCTCGAA SGC02 TAGGCCG SGC17 AGTC AGA SGC32 TACTCGC SGC03 GTCCGGC SGC18 TAGTCTA SGC33 GTACTAT SGC04 AGAATTA SGC19 CTCAGAT SGC34 TTGGATC SGC05 ATCCTCT SGC20 CCATACC SGC35 ACTTGCG SGC06 GAATCTC SGC21 ATAGCTG SGC36 CGC TATT SGC07 CCTAGGT SGC22 GACCGAT SGC37 ACTATCA SGC08 TGGAATA SGC23 CCTAACG SGC38 GACGTAC SGC09 CGCGCAG SGC24 TGCATGA SGC39 CGGACGT SGC10 ACGCGGA SGC25 AGGTACC SGC40 TGACGTC SGC11 GAGTAAC SGC...

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Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a multiple PCR method for improving amplification specificity and uniformity. The method comprises the steps as follows: extracting genome DNA from a sample; performing fragmentation and purification on the extracted genome DNA; carrying out a PCR amplification reaction by multiple specific primer pairs with the purified genome DNA as a template, wherein in the PCR amplification reaction, differential temperatures from low to high are used for annealing. The fragmentation and purification treatment is performed on DNA, the template and primers can be bond quickly, and the use rate of the primers is increased. Firstly, in a lower annealing temperature mode, amplification primers are fully bond to template DNA, the annealing temperature is increased gradually, and the binding specificity of all primers is improved; and amplification specificity and uniformity of the multiple PCR are remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a multiple PCR method for improving amplification specificity and uniformity. Background technique [0002] Multiplex PCR (multiplex PCR), also known as multiplex primer PCR or composite PCR, is improved on the basis of ordinary PCR, adding two or more pairs of specific primers to the same PCR reaction system, targeting multiple DNA templates or different DNA templates of the same template. PCR technology for regional amplification of multiple target fragments. Due to its high efficiency, systemicity, and economic simplicity, multiplex PCR has developed rapidly since it was proposed, and has now become an important application technology in the fields of life sciences. It is widely used in the simultaneous detection or identification of a variety of pathogenic microorganisms, the typing and identification of certain genetic diseases and cancer genes, e...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2537/143C12Q2527/101C12Q2525/191Y02A50/30
Inventor 朱碧银王益民何鑫玺王晓锋卜中鑫杜元平
Owner GENETALKS BIO TECH CHANGSHA CO LTD
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