ABO gene haploid typing method and reagent

Abstract

The invention relates to an ABO gene haploid typing method and a reagent. The ABO gene haploid typing method comprises the following steps: (1) designing and synthesizing a PCR amplification primer capable of expanding specific alleles of a growth fragment; (2) preparing human genome DNA; (3) according to the ABO genotype, carrying out selective PCR reaction to amplify the DNA sequence of the haploid part of the ABO gene in the human genome DNA; (4) carrying out double enzyme digestion purification on an amplification product; (5) providing oligonucleotide sequencing primers, and carrying outsequencing PCR on the double-enzyme-digested purified product; (6) purifying a sequencing product by a sodium acetate-ethanol precipitation method, and performing capillary electrophoresis sequencing;and (7) determining the ABO gene haploid and specifying the genotype of the ABO gene haploid. According to the ABO gene haploid typing method provided by the invention, haploid forms of three allelescan be selectively separated, and the method has wide application value in the aspects of ABO difficult blood type identification, ABO new allele sequence confirmation, accurate typing and the like.

Description

technical field [0001] The invention belongs to the technical field of genotyping detection methods, in particular to an ABO gene haploid typing method. The invention also relates to a typing reagent used in the ABO gene haploid typing method. Background technique [0002] Human ABO blood group is the most clinically significant blood group system, and its accurate typing is of great significance to clarify individual blood group genotype. [0003] ABO blood group antigens are determined by the ABO gene. The gene is located on human chromosome 9 (9q34.1-34.2) and is controlled by three multiple alleles of A, B and O. At present, the three alleles of A, B and O have been reported to have more than 400 gene forms containing different mutations, and new mutants are constantly being discovered. Since the human chromosome is diploid, the individual ABO blood group genotype usually exists as a single or a combination of two alleles among the three multiple alleles. The existin...

AI Technical Summary

Problems solved by technology

Therefore, when there is a new point mutation or the combination is not clear, the existing typing technology cannot determine the allele where the new mutation point is located, nor can it judge the ABO genotype of the individual, which can only be the result of speculation

However, such problems can only be solved by separating haploids, and obtaining ABO gene haploids can only be achieved by gene cloning technology to achieve the separation of two alleles.

However, there are two main problems in the monoclonal technology of ABO gene. First, the gene sequence of DNA clone is too long, which makes cloning difficult. However, due to the existence of transcription and splicing of ABO gene, it is difficult to obtain the cDNA sequence of cDNA sequence.

Second, the cloning technology itself is extremely cumbersome and takes a long time. It usually takes 3-4 days from cloning to sequence identification, and the cloning process can introduce false mutations. Therefore, the traditional cloning technology to obtain the ABO gene Haploid is currently more limited

Images