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Universal multiple PCR (Polymerase Chain Reaction) method

A multiplex and specific technology, applied in the field of general multiplex PCR, can solve the problems that need to be further improved, and achieve the effects of improving specificity and yield, shortening PCR time, and improving versatility

Inactive Publication Date: 2012-11-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been some research reports on improving the versatility of multiplex PCR, and some methods have been improved, but further improvement is needed

Method used

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  • Universal multiple PCR (Polymerase Chain Reaction) method
  • Universal multiple PCR (Polymerase Chain Reaction) method
  • Universal multiple PCR (Polymerase Chain Reaction) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 5

[0023] 1 material

[0024] Commercial corn seeds Zhengdan 958 and Xianyu 335.

[0025] 2 methods

[0026] (1) DNA extraction

[0027] After the dried corn seeds were ground, DNA was extracted by SDS method, and the extracted DNA was diluted to 100 ng / μL.

[0028] (2) Universal multiplex PCR primer screening

[0029] In this embodiment, 5 pairs of SSR primers with high polymorphism were selected (see Table 2 for primer parameters). Small multiplex PCR is easier to succeed), and after these 5 pairs of primers amplify the DNA of Zhengdan 958 and Xianyu 335, the sizes of the PCR products are different, so Zhengdan 958 and Xianyu 335 can be distinguished according to the size of the target band.

[0030] Table 2 Adapter and Primer Parameters

[0031]

[0032] universal adapter-F: the universal adapter of the upstream primer; universal adapter-R: the universal adapter of the downstream primer; phi085-F: the upstream primer; phi085-R: the downstream primer; U-phi085-F: the up...

Embodiment 2

[0042] 1 material

[0043] 200 commercial corn seeds of Zhengdan 958 were selected for purity testing.

[0044] 2 methods

[0045] (1) DNA extraction

[0046] After the single corn seed was ground, DNA was extracted by SDS method, and the extracted DNA was diluted to 100 ng / μL.

[0047] (2) Universal multiplex PCR primer screening

[0048] In order to illustrate the versatility of the multiplex PCR of the present invention, the adapter and primer parameters used this time are shown in Table 4, and the Tm value span of these 5 pairs of primers is 57.3~65.52 (when performing multiplex PCR, this span belongs to relatively large, span The smaller the multiplex PCR, the easier it is to succeed), and after the 5 pairs of primers amplify the DNA of Zhengdan 958, the size of the PCR product is different, so that the band pattern of Zhengdan 958 and the female parent of Zhengdan 958 can be distinguished according to the size of the target band. Banding of self-crossing grains and b...

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Abstract

The invention relates to a universal multiple PCR (Polymerase Chain Reaction) method. The universal multiple PCR method comprises the steps of: increasing a universal joint sequence to decrease difference of annealing temperature between primers, and increasing the annealing temperature (about 70 DEG C) of the jointed primers to be close to extension temperature; combining annealing and extensionphases; shortening PCR time; and improving the specificity and the yield of a PCR product by using two sections of circulation modes according to the gradual annealing circulation of primer annealingtemperatures from high to low. By using the universal multiple PCR method, the universality of multiple PCR can be remarkably improved, the universal multiple PCR method can be applied to many fields, such as seed purity identification, variety authenticity identification, polymorphism analysis, mutation analysis, quantitative analysis and species identification.

Description

1. Technical field [0001] The invention relates to a general multiplex PCR method, in particular to a primer design method for multiplex PCR technology and the establishment of a corresponding PCR amplification program, especially capable of improving the versatility of multiplex PCR. 2. Background technology [0002] Multiplex PCR (Multiplex PCR) is improved on the basis of ordinary PCR. Multiple pairs of specific primers are added to a PCR reaction system to amplify multiple target fragments for multiple DNA templates or different regions of the same template. Technology, this concept was first proposed by Chambercian J.S. et al. (1988). Multiplex PCR has the advantages of saving time, reducing costs, and improving efficiency. It has developed rapidly since it was proposed, and has been applied in many fields of life sciences (such as: polymorphism analysis, mutation analysis, quantitative analysis, and species identification, etc.) , has become an important application t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 张春庆温大兴
Owner SHANDONG AGRICULTURAL UNIVERSITY
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