Systemic displacement polymerase lymerase chain reaction

A polymerase chain reaction technology, which is applied in the field of system replacement polymerase chain reaction, can solve the problems of high false positives and easy PCR contamination.

Inactive Publication Date: 2007-10-31
北京万达因生物医学技术有限责任公司
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Problems solved by technology

[0004] In order to overcome the shortcomings of easy c

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

[0086] Embodiment. Hepatitis B HBsAg gene detection:

[0087] (1) Purification of DNA from 11 clinical specimens (according to Qiagen instructions):

[0088] 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.

[0089] 2): Transfer 100-200ul of the supernatant to a new EP tube, add an equal amount of chloroform, shake, and centrifuge.

[0090] 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.

[0091] (2) Indicates the preparation of template tailed DNA PCR:

[0092] 1) Select the gene sequence of hepatitis B virus HBs Ag aa124-138

[0093] 5’-gc acg att cct gct caa gga acc tct atg ttt ccc tct tg-3’

[0094] 3'-cg tgc taa ggt cga gtt cct tg-5' as the reserved sequ...

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Abstract

The invention discloses a 'system displacement polymerase lymerase chain reaction', which is characterized by the following: displacing program PCR system with above one indicating PCR system; setting the each indicating PCR system augment as small part crossing sequence of independence indicator board and program mold; possessing non-homologous sequence in indicator board with program mold; inverting the program specimen PCR to about ten non-interference indicated PCR system; changing to another system when polluted; avoiding twice pollution of PCR product; crossing the indicated DNA with program DNA; or crossing with program RNA; augmenting directly; fitting for direct test of RNA specimen; co-using with fluorescence molecular beacon (Molecular beacon); fitting for quantitative determination of the program mold.

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), which was made practical after many improvements by PE-Cetus. 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 a lot of PCR application technology and im...

Claims

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Application Information

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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
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