Method and kit for realizing sequencing-based typing of mitochondria 12S rRNA genome full-length sequence

Abstract

The invention belongs to the field of biological technical detection, and discloses a method and a kit for realizing sequencing-based typing of a mitochondria 12S rRNA genome full-length sequence. The method comprises the following steps: carrying out PCR amplification reaction on a typing target area, namely a 954bp genome full-length sequence, by adopting a pair of PCR amplification primers, and carrying out bi-directional sequencing reaction on the amplification product through four forward and reverse sequencing primers. The invention establishes the sequencing-based typing method for the 12S rRNA genome full-length sequence which is accurate, stable and reliable. The obtained base sequence peak map contains no background signals and impure peaks, and is easy for identification and result determination. The method can carry out sequencing-based typing on the 12S rRNA genome full-length sequence, so that the equivocal result appearing in the sequencing-based typing is avoided; the method is suitable for the typing of the 12S rRNA genes, and the basic and applied study works on the aspects of population genetics, evolutiology, disease association and the like of the 12S rRNA genes.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and relates to a method and a kit for sequencing and typing the full-length sequence of mitochondrial 12SrRNA genome. Background technique [0002] Since Prezant et al first discovered the A1555G mutation of the mitochondrial 12SrRNA gene in a non-syndromic maternally inherited Arab-Israeli deafness family in 1993, the mitochondrial 12SrRNA gene mutation has been confirmed in families of different countries, different races, and sporadic deafness patients It is related to aminoglycoside-induced deafness and non-syndromic deafness. As of February 2015, the NCBI database has published 36 mitochondrial 12SrRNA gene single nucleotide polymorphism (SNPs) sites, of which 7 sites are related to aminoglycoside drug-induced deafness or / and non-syndromic deafness Related, including: A827G, T961G, T1095C, T1291C, C1494T, A1555G and G1598A. A1555G and C1494T are the main sites of drug-induced deafnes...

AI Technical Summary

Problems solved by technology

Currently reported 12SrRNA fluorescence detection kits or restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) can only identify part of the 12SrRNA SNPs loci (C1494T and A1555G), and cannot achieve fine allele level and screening All variants limit the application of 12SrRNA at the allele level in the research and clinical application of population genetics, deafness and other diseases

Secondly, there is no recognized 12SrRNA sequencing typing method in the world so far. The reported methods have the following problems: (1) only part of the gene sequence is directly sequenced and typed, and the sequencing type does not cover the full-length sequence of the 12SrRNA genome. It is not conducive to the study of the polymorphism and its function of the full-length sequence of the 12SrRNA genome, and it is prone to ambiguous sequencing typing results

(2) Although the target typing fragment amplified by PCR during sequencing and typing covers the full-length sequence of the genome, multiple product fragments need to be amplified, and the experiment is time-consuming and laborious.

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