Method for targeted capture and sequencing of HLA gene sequences

Abstract

The invention relates to the field of biotechnology, and particularly provides a method for targeted capture and sequencing of HLA gene sequences. The method comprises the following steps: 1) hybridizing a denatured nucleic acid sample with a nucleic acid probe library fixed on a solid-phase carrier under a hybridization condition to form a target nucleic acid molecule-probe-solid-phase carrier membrane complex; 2) cleaning the 'probe-solid-phase carrier membrane complex' bound with target nucleic acid in step 1) by using a cleaning solution, eluting the probe-solid-phase carrier membrane complex bound with the target nucleic acid from a solid-phase carrier membrane by using an eluting solution, purifying, enriching or constructing to obtain a nucleic acid library; and 3) taking the nucleic acid library obtained by enrichment or constructed after enrichment in step 2) for high-throughput sequencing. A nucleic acid probe which can rapidly generate high-fold coverage and single-base displacement and is used for enriching HLA genes can be immovably fixed on the solid-phase carrier membrane. The method has characteristics of simplicity and convenience in operation, flexible probe acquisition and low cost, is favorable for improving the HLA genotype identification accuracy, and is rapid and convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, and specifically provides a method for targeted capture and sequencing of HLA gene sequences. Background technique [0002] Human Leucocyte Antigen (Human Leucocyte Antigen, HLA), also known as Major Histocompatibility Complex (MHC), this complex is the most complex human gene system known so far, located on the short arm of chromosome 6 , about 4000Kb in length, containing 224 loci, dozens of loci, and co-dominant expression, is the most complex genetic polymorphism system known to the human body. At the same time, each gene locus has dozens of alleles, such as A gene, B gene, and C gene related to immune rejection in the HLA-I family, which contain 3657, 4459, and 3290 alleles, respectively. HLA is expressed on the surface of human cell membranes and represents the antigen specificity of each individual. Therefore, HLA is closely related to the body's immune system, and the main cause of immune r...

AI Technical Summary

Problems solved by technology

From the 1970s to the end of the 1980s, serological identification methods with low resolution and accuracy were mainly used

However, similar to the HLA gene, it accounts for less than 0.1% of the whole human genome, and the target only accounts for a small part of the whole. Although human whole genome sequencing can achieve HLA typing, the required sequencing depth is high and the cost is huge

In this method, the method of magnetic bead capture requires artificial synthesis of probe sequences, which is costly

Secondly, the region targeted by the probe is an HLA non-coding region sequence, rather than a coding region sequence with typing information directly, which will result in a low proportion of coding regions, most of which are non-coding region sequences without information, resulting in waste

[0012] At present, the enrichment of HLA genes and high-throughput sequencing are carried out using solid-phase carrier membranes such as nylon membranes, cellulose acetate membranes, or similar, and there are no reports and studies on this aspect.

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