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Systemic displacement polymerase lymerase chain reaction

A polymerase chain and ligase reaction technology, applied in the direction of fermentation, can solve the problems of easy contamination and false positives in PCR

Inactive Publication Date: 2009-10-28
北京万达因生物医学技术有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the shortcomings of easy contamination and high false positives in PCR detection

Method used

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  • Systemic displacement polymerase lymerase chain reaction
  • Systemic displacement polymerase lymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0090] Embodiment. Hepatitis B HBsAg gene detection:

[0091] (1) Purification of DNA from 11 clinical specimens (according to Qiagen instructions):

[0092] 1): Add 100-200ul sample to a 1.5ml EP tube, add an equal amount of phenol / chloroform / isoamyl alcohol (25:24:1) for extraction, vortex, and centrifuge at the highest speed for 5 minutes in a desktop centrifuge.

[0093] 2): Transfer 100-200ul of the supernatant to a new EP tube, add an equal amount of chloroform, shake, and centrifuge.

[0094] 3): Add 4 times Qiagen binding buffer to the supernatant and transfer to the purification column, wash the column twice with the washing buffer, add 50ul dH 2 Purified samples were collected by O elution.

[0095] (2) Preparation of indicating template plus head-to-tail DNA PCR:

[0096] 1) Select the gene sequence of hepatitis B virus HBs Ag aa124-138

[0097] 5’-gc acg att cct gct caa gga acc tct atg ttt ccc tct tg-3’

[0098] 3'-cg tgc taa ggt cga gtt cct tg-5'

[0099] as...

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Abstract

The present invention relates to a "system displacement polymerase chain reaction", which is characterized in that the PCR amplification to be tested is replaced by the amplification of more than one set of indicator PCR systems through the template to be tested - indicator template hybridization ligase reaction, including the following Steps, (1) add part of the reserved sequence of the gene of the template to be tested to the two ends of the indicator template that has nothing to do with the template to be tested to form the indicator template plus the head and tail DNA; Complementary hybridization, (3) connect the head and tail gaps of the indicator template through T4 or Taq ligase reaction to form a circular indicator template; (4) amplify the circular indicator template by reverse PCR. The template to be tested can be either a DNA or an RNA sample, and due to the hybridization matching-dependent repair amplification, it is suitable for the detection of genetic mutations and genetic diseases, and it is also suitable for the quantitative determination of the template to be tested when used in conjunction with fluorescent molecular beacons.

Description

technical field [0001] The invention relates to the further development of polymerase chain reaction (PCR) gene amplification technology, a new PCR approach for molecular detection application, especially the replacement of gene detection PCR system with dozens of independent DNA indicator PCR systems for use in rotation. Background technique [0002] One night in 1983, K.B.Mullis inadvertently created the fantastic idea of ​​PCR (The unusual origin of the polymerase chain reaction, Sci, Am.262:56, 1990 and US patent#4683202), and approved by PE-Cetus company Many improvements and practical. With the application of thermostable DNA polymerase derived from thermophilic bacteria (Saiki R.K., et.al, Science 239:487-491, 1988), PCR technology has gradually become mature, efficient and automatic. It is widely used in gene cloning, sequencing, molecular detection and many other fields, laying a solid foundation for modern molecular biology. And gradually derived many PCR applica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 江洪江必胜廖同兵
Owner 北京万达因生物医学技术有限责任公司