Polymerase chain reaction-sequence based typing (PCR-SBT) method for ABO blood type genotyping and reagent

Abstract

The invention provides a polymerase chain reaction-sequence based typing (PCR-SBT) method for ABO blood type genotyping. The method comprises the following steps of: preparing human genome DNA; amplifying segments of ABO gene exon 1, exons 2-4 and exons 5-7; performing double enzyme digestion purification on the obtained amplified products; performing a sequencing PCR reaction on the purified products; purifying the sequenced products by a sodium acetate-ethanol precipitation method and performing capillary electrophoresis sequencing; and analyzing the obtained sequences by using software to determine the genotype. The method has the advantages of solving the problems of identification of an ABO subtype, judgment of difficult blood types, discovery of a new mutational site, gene recombination among genes, genetic polymorphism detection and the like, exerting the characteristics of high flux and result accuracy of ABO genotyping operation by PCR-SBT, achieving great importance for the relative application in the fields of clinical transfusion medicinal research, genetics and the like and having important practical significance for medicinal research units, pharmic research and reagent development units.

Description

technical field [0001] The invention relates to a genotyping detection method, in particular to a molecular biology detection method for ABO blood type genotyping, and also relates to reagents used in the method. Background technique [0002] The human ABO gene is located on chromosome 9 (9q34.1-34.2), controlled by three multiple alleles A, B and O, including 7 exons ranging in length from 28 to 688 bp and a length of about 19514 bp 6 introns, the total length is about 18 ~ 20kb. The gene coding product is a glycosyltransferase, most of the coding sequence (823bp in 1062bp) is located on the 6th and 7th exon, which is responsible for encoding the catalytic region of the glycosyltransferase. These transferases control the biosynthesis of ABO blood group antigens, thereby determining their blood type. The DNA sequences of other alleles of ABO blood type are highly conserved and closely related to the DNA sequence of A101, with only a few base substitutions or single base de...

AI Technical Summary

Problems solved by technology

However, the current ABO blood group PCR-SBT typing method mainly focuses on the determination of exons 6 and 7, which are relatively concentrated polymorphic sites, to determine its genotype, and does not sequence exons 1-5, especially It is exon 1 containing the start codon, its GC content is relatively high, and PCR amplification is difficult to obtain. This operation method has two defects: first, due to the single nucleotide polymorphism of exons 6 and 7 The sex is very concentrated, and the difference between each ABO blood type may only be caused by a single or a few nucleotide variations, which may easily cause missed detection of SNP sites (single nucleotide polymorphism sites), causing blood group gene type of misjudgment

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