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Method for PCR primer design for sequence-unknown genes

A primer design and gene technology, applied in the field of biological PCR detection, can solve the problems of extremely high operating technology and equipment requirements, low abundance of high-level structures, time-consuming and labor-intensive problems, and achieve the effect of simple and feasible technology, simplified labor, and wide application range

Inactive Publication Date: 2012-03-28
FUDAN UNIV
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Problems solved by technology

The prerequisite for the success of RACE is that the reaction of mRNA reverse transcription into cDNA sequence should be well controlled, but because mRNA is easy to degrade, the reverse transcription reaction is particularly vulnerable to low abundance and high-order structure
However, the method of using isotope or biotin-labeled probes to hybridize and screen cDNA libraries is more time-consuming and laborious, and it is the last choice for researchers when they have to.
The above traditional methods require extremely high operating techniques and equipment, and are expensive, which are not available in general laboratories.

Method used

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Embodiment Construction

[0018] In the process of scientific research, we need to detect the expression of DNA-PKcs mRNA, a gene involved in DNA repair in Chinese hamsters, but since the Chinese hamster DNA-PKcs mRNA cannot find the corresponding sequence on Genebank, we use this invention to design primers and sequence Unknown Chinese hamster DNA-PKcs gene was detected by PCR.

[0019] (1) Cross-species homology comparison method to design PCR primers

[0020] 1. Through BLAST nucleotide sequence comparison, find the conserved sequence of DNA-PKcs mRNA between mice and humans. The sequence shown in the table below is the most conserved sequence of DNA-PKcs mRNA between mice and humans. 84%, Query is the DNA-PKcs mRNA sequence of mice, and Sujct is the DNA-PKcs mRNA sequence of humans;

[0021]

[0022] 2. Use the primer design software primer5.0 to design the upstream and downstream primers respectively based on the conserved sequences. The following table shows the primer sequences for PCR;

[...

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Abstract

The present invention belongs to the technical field of biological PCR detection, particularly a method for PCR primer design for sequence-unknown genes. By the principle that identical genes have conserved sequences across different species, the method performs nucleotide sequence alignment through BLAST software, so as to find the conserved sequence of a sequence-unknown gene and accordingly design a primer for PCR detection on the sequence-unknown gene. The present invention solves the problems of complex operation and high cost when PCR detection is performed on a sequence-unknown gene with the traditional method. In accordance with the cross-species PCR primer design method of the present invention, a sequence-unknown Chinese hamster gene is amplified successfully. The present invention has the advantages of simple feasible technical design, fewer operation limitations, wide application range, reliable results and low cost.

Description

technical field [0001] The invention belongs to the technical field of biological PCR detection, and relates to a method for performing PCR detection on genes with unknown sequences by using bioinformatics and PCR technology to design primers from the conserved sequences of genes with known sequences. Background technique [0002] PCR (Polymerase Chain Reaction) technology is an in vitro enzymatic reaction that specifically amplifies DNA. It can amplify DNA with a known sequence in a short time and is used for detection of expression, diagnosis, signing, and preparation of probes. It is a An extremely effective and practical technique. The characteristics of PCR show that if the DNA or mRNA sequence of a gene is not known, it is impossible to conduct in-depth research on the structure and function of the gene. At present, most of the gene sequences of common species such as humans and mice are already clear, but some experiments Most of the genes of species commonly used in...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 邵春林袁德晓潘燕张江虹沈波
Owner FUDAN UNIV
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