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PCR method for overcoming lagging pollution

A lagging, polymerase technology, applied in the field of molecular biology, which can solve the problems of complex operation and inability to clone the product.

Inactive Publication Date: 2005-02-23
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a PCR method for overcoming hysteresis pollution, to fill in the gaps in solving the problem of hysteresis pollution through primer design and conventional restriction endonuclease treatment at present, and to solve the need for special reagents, enzymes, and operations to overcome hysteresis pollution in the prior art. Complicated, the defect that the product cannot be cloned

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Use a pair of primers to amplify a fragment of the GPT-binding protein RhoA gene (gene bank number L25080), and then dilute the PCR amplification product as the amplification template for the next round of PCR reaction. Before the new round of PCR reaction, use one restriction endonuclease or two restriction endonucleases that have a cutting site for the fragment sequence, and use the enzyme that has been completely inactivated by heating as a control. According to the cycle number of the first appearance of the new amplified product band, the degree of damage to the substrate DNA by the restriction endonuclease is estimated. The sequence of forward and reverse primers is shown in Table 3.

[0036] name

[0037]The length of the amplified product is 590bp, and the restriction endonucleases EcoRV and PvuII each have a cutting point for the amplified product fragment. First, commercial human tissue total RNA was used as the original template, and cDNA...

Embodiment 2

[0042] In Example 2, a similar analysis was performed using the human beta-2-adrenoceptor gene (GenBank No. M15169) and human glyceraldehyde-3-phosphate-dehydrogenase gene (GenBank No. XM_006959) as examples. The primer sequence is shown in Table 5.

[0043] name

Location

Sequential (5' to 3')

BAR positive

1574-

1589

CCA GAC TGC GCG CCA T

BAR reverse

1805-

1825

GAT CAG CAC AGG CCA GTG AAG

GPH Positive

553-591

CTG GCC AAG GTC ATC CAT GAC AAC TTT

GGT ATC GTG GAA

GPH reverse

904-940

CGC TGT TGA AGT CAG AGG AGA CCA

CCT GGT GCT CAG T

[0044] The amplified product of the adrenoceptor gene is 252 bases in length, and is simultaneously treated with two kinds of restriction endonucleases PstI and HaeII; The enzymes XhaI and HaeII act. cDNA preparation, first round and second round ...

Embodiment 3

[0047] Example 3 A pair of primers were designed with the human actomyosin gene as the target gene (GenBank No. BC016045). The forward and reverse primers matched the sequence of the target gene, but a mismatched base was introduced respectively, and the forward primer caused an EcoRI cleavage site , while the antiprimer creates a HindIII cleavage site. The sequences and properties of the primers used in Example 3 are shown in Tables 7 and 8.

[0048] name

[0049] The italicized bases in the primers "A forward" and "A reverse" are the introduced mismatched bases, and the underlined parts are the recognition sequences of EcoRI and HindIII formed after the introduction of the mismatched bases. Primer "A1 positive" is the same as the 3' end of primer "A positive"; primer "A1 anti" is the same as the 3' end of primer "A anti".

[0050]

[0051] Oligo(dT) was used to synthesize cDNA, and the reagents and methods for amplifying cDNA with primer "A" were the sam...

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PUM

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Abstract

A PCR method for overcoming hysteresis pollution is disclosed. It achieves simple process and good effect.

Description

technical field [0001] The present invention relates to molecular biology techniques, in particular to polymerase chain reaction methods. technical background [0002] Polymerase chain reaction (PCR) is a fast and efficient method for amplifying specific DNA. According to the basic principle of PCR, after 25-30 cycles, the target gene fragment will be increased by 8 million to 250 million times. Since both the amplified product and the target gene in the sample can be used as amplification templates, the delayed contamination of traces or even a few copies of the amplified product DNA fragment will cause much more trouble than cross-contamination. In order to reduce the chance of delayed contamination, strict physical precautions are usually taken (J.L.Hartley et al., PCR Method and applications, 1993, 3, S10-S14), such as separating the PCR reaction solution preparation area from the PCR product detection area or even dividing them into two rooms , use a special PCR opera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
Inventor 徐定邦朱德芬谢文凯徐文慧
Owner 徐定邦
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