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HLA gene specific PCR amplification primer, HLA typing method and kit

A technology of amplification primers and genotyping, which is applied in biochemical equipment and methods, DNA/RNA fragments, and microbial determination/inspection. It can solve problems such as low resolution and inability to detect alleles. The effect of image quality improvement, reasonable and accurate judgment, and reliable data

Inactive Publication Date: 2014-03-12
SHANGHAI TISSUEBANK BIOTECH +3
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Problems solved by technology

However, the disadvantage of this method is that it cannot detect new alleles, the resolution is not high, and the detection signal is an analog signal

Method used

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  • HLA gene specific PCR amplification primer, HLA typing method and kit
  • HLA gene specific PCR amplification primer, HLA typing method and kit
  • HLA gene specific PCR amplification primer, HLA typing method and kit

Examples

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Embodiment 1

[0070] Example 1 Carrying out HLA typing detection on samples of known genotypes

[0071] In this implementation, PCR amplification primer pairs and sequencing primers as shown in Table 1 were used to conduct HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA - High-resolution genotyping confirmation of the DQB1 locus. The specific steps of the test are as follows:

[0072] 1. Genomic DNA extraction

[0073] (1) Draw 600μl of mixed whole blood into a labeled 2ml EP tube;

[0074] (2) Add 1000 μl red blood cell lysate (Beiyuntian Biotechnology Research Institute, Cat. No. C3702), shake well until the mixture is bright, and centrifuge at 11000 rpm for 1 min;

[0075] (3) Discard the supernatant, buckle the opening of the EP tube downward on the absorbent paper, and drain the remaining residual liquid as much as possible;

[0076] (4) Add 1000 μl red blood cell lysate, repeat (2);

[0077] (5) Discard the supernatant, put the opening of the EP tube downward on the absorbent paper, and drain...

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Abstract

The invention discloses an HLA (human leukocyte antigen) gene specific PCR (polymerase chain reaction) amplification primer, an HLA typing method and an HLA typing kit. A sequence of the PCR amplification primer comprises a primer sequences shown as SEQ ID No. (sequence identification number) 1-2, 3-4, 5-6, 7-8 and 9-10. The HLA gene typing method comprises the steps that the HLA gene specific PCR amplification primer is used for conducting PCR amplification and purification on sample DNA (deoxyribonucleic acid); a gene exon sequencing primer is used for conducting sequencing PCR amplification, purification and sequencing on an HLA gene amplification product, the sequence of the HLA gene amplification product is compared with a standard sequence; and the type of the HLA gene is determined. The HLA gene typing kit comprises the PCR amplification primer and the sequencing primer. A qualitative leap in aspects of detection flux, data quality, cost control and the like is achieved; data is more reliable and realer; and the problem that typing cannot be conducted when nucleotide of certain alleles is positioned outside an amplification area is solved.

Description

technical field [0001] The invention relates to a gene amplification primer and a method and kit for genotyping using the primer, in particular to a HLA (human leukocyte antigen) gene-specific PCR amplification primer and using the primer for HLA typing methods and kits. Background technique [0002] The Human Leukocyte Antigen (HLA) gene is located on the short arm of human chromosome 6 and is the most polymorphic gene found in humans. The HLA genes are divided into three classes: class I, class II and class III genes. Among them, class I includes: HLA-A, HLA-B, HLA-C genes; class II includes: HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1 genes. [0003] The HLA system is the most complex polymorphic system known to the human body. Since the discovery of the first HLA antigen (Jean Dausset) in 1958, until the 1970s, HLA has become an important emerging research field in the disciplines of immunogenetics, immunobiology and biochemistry. Now, the composition, structure...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 郑仲征黄锟潘捷邢宽龙飞艳卓孝福王宁娟
Owner SHANGHAI TISSUEBANK BIOTECH
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