A method and application for mining low-abundance expressed genes and systematically identifying splice variants using nested PCR combined with gene-specific primers for reverse transcription

A technology for expressing genes and specific primers, applied in the field of biotechnology applications, can solve the problems of high false positives, high cost, no mining of low-abundance expressed genes, and systematic identification of splice variants.

Inactive Publication Date: 2011-11-30
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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Problems solved by technology

Although these technologies can obtain low-abundance expression genes to a certain extent, they have their own shortcomings, such as high false positives, poor stability, cumbersome steps, heavy workload, long cycle, and high cost, making it difficult to popularize Therefore, there is currently no effective method for mining low-abundance expressed genes and systematically identifying splice variants

Method used

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  • A method and application for mining low-abundance expressed genes and systematically identifying splice variants using nested PCR combined with gene-specific primers for reverse transcription
  • A method and application for mining low-abundance expressed genes and systematically identifying splice variants using nested PCR combined with gene-specific primers for reverse transcription
  • A method and application for mining low-abundance expressed genes and systematically identifying splice variants using nested PCR combined with gene-specific primers for reverse transcription

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Design of gene-specific primers (GSP) for mining low-abundance expressed genes and systematically identifying splice variants

[0042] The design of gene-specific primers (GSP) is automatically completed by the Primer3 program, and the main idea of ​​the program design is as follows:

[0043] 5) Obtain the sequence of a gene splicing variant from the NCBI GenBank database, and analyze its gene structure according to the GenBank database records or using software such as splign, gmap, blat, blast;

[0044] 6) Search for possible candidate primer regions within the full-length cDNA sequence;

[0045] 7) Scoring the candidate primers, which consists of three parts, namely S=S basic +S end +S genome :

[0046] a) Primer quality basic score (S basic ): Scored according to the conventional primer quality evaluation system, that is, the primer length is 18-25bp, the GC content is about 50%, and the Tm is about 58°C. The closer to these parameters, the higher th...

Embodiment 2

[0060] Example 2: Application of gene-specific primers (GSP) in combination with nested PCR technology in identification and discovery of low-abundance expression genes

[0061] Using gene-specific primers (GSP, taking the primers designed in Example 1 as an example) in combination with the application of nested PCR technology in the identification and discovery of low-abundance expression genes, the specific method includes the following steps (the flow chart is as follows: figure 1 shown):

[0062] 1. 3’GSP1-dependent RNA reverse transcription

[0063] Extracted total RNA (wild-type Caenorhabditis elegans N 2 strain or mouse) as a template, the first strand of cDNA of the corresponding gene was reverse transcribed. The 20 μl reverse transcription system and reverse transcription method based on 3’GSP1 are as follows (the reagents used are purchased from TOYOBO): (1). Total RNA 1 μg, corresponding gene 10 pmol / L 3’GSP1 1 μl, RNase Free H 2 O make up to 12μl, mix well, 65°C...

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Abstract

The invention discloses a method for excavating a low-abundance expression gene and system identification splicing variants by using a reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof. The method is based on reverse transcription PCR of 3'GSP1, gradient PCR is utilized to search for the best annealing temperature of each gene for each primer pair combination, and whether it is a new splicing variant is judged on the basis of GSP nested PCR sequencing and sequence analysis. The method is simple and easy to implement, has the advantages of favorable repetitiveness, high stability and strong reliability, greatly saves the experimental cost, can be applied to excavation of eucaryote low-abundance expression gene under different physiopathological states, and identification and detection of high / low-abundance expression gene splicing variants, and can be used for excavating low-abundance expression gene and identifying and detecting splicing variants of high expression gene and low-abundance expression gene. The method can be used for detecting both known splicing variants of targeted genes and unknown splicing variants of genes, and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology applications, in particular to a method for mining low-abundance expressed genes and systematically identifying splice variants using nested PCR combined with gene-specific primer reverse transcription technology, which can be applied in different environments for low Abundantly expressed genes and the expression of alternatively spliced ​​variants are being studied and evaluated. Background technique [0002] Studies have reported that about 95% of genes in higher eukaryotes undergo alternative splicing. Genes express specific splicing forms in specific environments, and the latter are often biomarkers of certain diseases, and may also be the cause of these diseases. Therefore, it is important to discover as many alternative splicing forms of important genes as possible in biological and medical research aspect is significant. However, due to the abnormally low abundance of transcripts of some gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11G06F19/28C12N15/10C12Q1/68C12N15/12
Inventor 张成岗张艳春严瑞芬屈武斌吴永红高艳
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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