Polymerase chain reaction method for overcoming hysteresis pollution using limiting endonuclease

A technology of restriction endonuclease and endonuclease, applied in the field of molecular biology, can solve the problem that non-natural nucleotide substrates and amplification products cannot be cloned, etc.

Inactive Publication Date: 2006-01-11
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention provides a PCR method for overcoming hysteresis pollution, to fill in the current lack of solving the problem of hysteresis pollution through primer design and specific restriction endonuclease methods, and to solve the need to use non-natural nucleotides in existing techniques for overcoming hysteresis pollution Substrates and amplified products cannot be cloned

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Introduce a restriction endonuclease recognition sequence in the forward primer, and the target genes are human beta-2 adrenoceptor gene (National Center for Biological Information Gene Bank number M15169) and human glyceraldehyde-3-phosphate dehydrogenase respectively Gene (National Center for Bioinformatics GenBank No. XM_006959). Primer sequences and properties are shown in Tables 2 and 3. The bold font is the recognition sequence of the restriction endonuclease, the bold font and the underline are the introduced mismatched bases, and the parts separated by the underline are the cutting sites of the endonuclease.

[0026] Primer

[0027]

[0028] It can be seen that after the restriction endonuclease recognition sequence is introduced into the 5' end of the forward primer, the number of matching bases between the forward primer and the amplification product decomposed by the endonuclease is less than 10, and it is impossible to reconci...

Embodiment 2

[0030] Embodiment 2 respectively introduces two restriction endonuclease recognition sequences in forward primer and antiprimer, and target gene is human beta actomyosin gene (numbering BC016045 of U.S. National Biological Information Center Gene Bank), primer sequence and characteristic are shown in Tables 4 and 5. The bold font is the recognition sequence of the restriction endonuclease, the bold font and underline are the introduced mismatched bases, and the parts separated by the underline are the cutting sites of the restriction endonuclease.

[0031] Primer

[0032] lead

[0033] Human genomic DNA was used as a template to amplify with the Advantage-2-PCR kit of Clontech Company. The reaction volume of each tube is 25 microliters, and the primer concentration is 0.4uM. The PCR program is: first denaturation at 94°C for 1 minute, cycle denaturation at 94°C for 10 seconds, annealing and extension at 70°C for 1.5 minutes, 32 cycles in total. After the...

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PUM

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Abstract

A PCR method for preventing the hysteresis pollution by using endodeoxyribonuclease features that the mispairing base is introduced to the terminal 5' of primer for configuring the recognition sequence of endodeoxyribonuclease in the amplified product in such manner that the position of cutting nucleic acid by endonuclease is the 4-20 bases at the upstream of said recognition sequence.

Description

technical field [0001] The present invention relates to molecular biology techniques, in particular to a polymerase chain reaction method for preventing hysteresis contamination. technical background [0002] Polymerase chain reaction (PCR) is a fast and efficient method for amplifying specific DNA. After about 30 cycles, the target gene fragment is amplified hundreds of millions of times. Since both the amplified product and the original target gene in the sample can be used as templates for amplification, traces of PCR reaction solution hysteresis contamination will cause much more trouble than sample cross-contamination. Methods to overcome hysteresis contamination include physical precautions (J.L. Hartley et al., PCR-Methods and-applications, 1993, 3: S10-S14) and various chemical and biochemical methods. M.C. Longo et al. (Gene, 1990, 93:125-128) used dUTP to replace the natural nucleotide dTTP as the substrate of the reaction, so that the lagging amplification produc...

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 徐定邦朱德芬谢文凯
Owner 徐定邦
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