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High variation zone amplication primer of rockfish mitochondrial genome and its design method

A technology of mitochondrial genome and amplification primers, which is applied in the field of PCR primers to achieve rapid and accurate identification

Inactive Publication Date: 2010-08-04
XIAMEN UNIV
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Problems solved by technology

However, the sequences at both ends of the mitochondrial genome hypervariable region of the grouper are more specialized, and the reported amplification primers for the mitochondrial genome hypervariable regions of other species, such as L15926 (Kocher et al., 1989), H16498 (Meyer et al., 1990 ), L16518 (Meyer), etc. cannot effectively amplify it

Method used

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Embodiment Construction

[0019] The grouper mitochondrial genome hypervariable region amplification primer (G-dloop) of the present invention is made up of two single-stranded oligonucleotides, wherein the light chain primer G-dloop (L) has 22 bases: CAG AGC GCC GGT CTT GTA AACC, located on the tRNA-Thr gene, the heavy chain primer G-dloop (H) has 21 bases: GTC AGG ACC AA(A / G)CCT TTG TGC, one of which is a degenerate base , located at the 3' start of the 12SrRNA gene.

[0020] The above primers were used for PCR amplification of 32 species of grouper, all of which could obtain specific amplification products with fragment sizes ranging from 1000 to 1200 bp. After sequencing and comparison with homologous sequences on Genbank, it was confirmed that they contained high variation in the mitochondrial genome Amplified products of the full sequence of the region. Therefore, it provides a powerful tool for the species identification, geographical population identification and germplasm resource evaluation ...

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Abstract

The present invention relates to one pair of PCR primers named as G-dloop, and provides one pair of amplification primers capable of amplifying the high variation zone of rockfish mitochondrial genome in high efficiency and its design method. The amplification primers consist of two single strand oligonucleotides. Through logging-on Genebank to search vertebrate mitochondrion DNA cytochrome b gene and the high conservation area of 16S rRNA gene sequence and homologous comparison, one pair of amplification primers is obtained. Through long PCR amplification, the target segments of 32 varietiesof rockfish are obtained and sequenced. The obtained sequences are compared by means of using homologous comparison software Clustal X 1.83 to fine the conservation sequences in the cytochrome b5' ends and the 12S rRNA 3' ends of the mitochondrial genomes of the 32 varieties of rockfish, and the said amplification primers are designed based on the degeneracy principle.

Description

technical field [0001] The invention relates to a pair of PCR primers (named as G-dloop), in particular to an amplification primer mainly used for specifically amplifying the highly variable regions (ie, control regions) of mitochondrial genomes of most grouper fishes. Background technique [0002] Because animal mitochondrial DNA (mtDNA) has the characteristics of simple structure, strict maternal inheritance, almost no recombination, fast evolution rate and differences in evolution rate in different regions, it is widely used in the research of species identification and taxonomic status. The mitochondrial genome consists of 37 coding genes and a major non-coding region (control region), and the evolution rates of different sequence regions are different. Among them, the fastest evolution rate is the control area, which is suitable for the comparative study among groups with close kinship. [0003] At present, it is a very convenient and accurate method to identify fish w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 丁少雄杜佳莹王军苏永全
Owner XIAMEN UNIV
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