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PCR method capable of eliminating non specific braid and RT-PCR chip prepared using said method

A non-specific, banded technology, applied in fermentation and other directions, to achieve the effect of easy promotion

Inactive Publication Date: 2008-11-12
NANHUA UNIV
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Problems solved by technology

Therefore, the primer extension reaction mediated by Pfu high-fidelity DNA polymerase alone will also appear non-specific bands, which is also a defect that this type of high-fidelity DNA polymerase cannot be widely used

Method used

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  • PCR method capable of eliminating non specific braid and RT-PCR chip prepared using said method

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Embodiment Construction

[0016] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0017] Gaveolin-1 gene is amplified by using the present invention. In Figure 1, 3(M) represents Marker, and bands 1 and 2 represent the full length and fragment of the gene Gaveolin-1, respectively.

[0018] Specific steps are as follows:

[0019] The first step: the extraction of mouse Gaveolin-1 (caveolin 1) mRNA and the synthesis of cDNA are carried out according to conventional methods;

[0020] The second step: primer design. This embodiment designs four kinds of primers:

[0021] Primer 1: 3' end mismatched and unsulfurized primer (bands 14-15, 18);

[0022] Primer II: 3'-end mismatched and sulfur-modified primers (bands 4-9);

[0023] Primer III: 3'-end matching primers without sulfur modification (bands 12-13);

[0024] Primer IV: 3'-end matched and sulfur-modified primers (lanes 1-2, 10-11, 16-17).

[0025] The third step: the ...

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Abstract

A PCR method for eliminating the non-specific band and precisely obtaining target gene by one step, and the RT-PCR chip prepared by said method are disclosed. When the primer of (PCR CRT-PCR or PCR) target gene is designed, the one or more bases at terminal 3' of primer are modified by thiophosphate. The polymerase used for extension reaction is the DNA polymerase with high Pfu fidelity. As a result, in various PCR reactions, only the template fully matched with 8bp at terminal 3' of primer can be extended with high fidelity to said template.

Description

technical field [0001] The present invention relates to a PCR (RT-PCR, PCR) method suitable for chip technology to obtain specific gene expression (including coding region, transcriptome, expression profile comparison), diagnosis and RT-PCR chip prepared by the method, especially A PCR method that can eliminate non-specific bands and accurately obtain target genes in one step to build a technical platform for obtaining specific gene expression (including coding regions, transcriptomes, and expression profiles) and diagnosis with high precision and high throughput. Background technique [0002] With the completion of the draft human genome DNA sequence ahead of schedule, human beings have entered the post-genome era. In the post-genome era, it is first required to use human genome DNA sequence information to study the structure and function of genes and their protein products, and further to their functional mechanisms in the occurrence, development, aging, death, and normal ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 杨惠龄廖端芳徐阳炎罗迪贤李凯
Owner NANHUA UNIV
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