Liquid-phase gene chip system for human leukocyte antigen gene typing
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A human leukocyte antigen and genotyping technology, applied in the field of biomedicine, can solve the problems of inability to achieve high throughput, lack of stability, and specificity
Inactive Publication Date: 2007-04-25
上海复旦张江生物医药股份有限公司
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Problems solved by technology
[0008] Another object of the present invention is to provide a gene chip composed of the above-mentioned probe and carrier, to overcome the problems that the prior art cannot realize high throughput and insufficient stability, repeatability and specificity;
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Embodiment 1
[0090] Embodiment 1, the extraction of genomic DNA
[0091] A4008 A4009 A4010 A4011 A4012 A4013 blood sample, use the DNA extraction kit of Shanghai Sangong Bioengineering Technology Service Company to extract genomic DNA, the concentration is 20 ~ 100ng / ul, OD 260 / 280 =1.5~1.8.
Embodiment 2
[0092] Embodiment two, the PCR amplification of the second and third exon of HLA-A gene
[0093] Prepare 10X buffer: 100mM Tris-HCl, pH9.0, 100mM KCl, 80mM (NH 4 ) 2 SO 4 , 0.5% NP-40, 20mM MgSO 4 , 0.22um membrane filtration. Glycerin plus double distilled water, 121°C, 15 minutes, autoclave for later use.
[0094] Preparation of P Mix solution: primers AE2F1, AE2R2, AE2R1, AE3F1, AE3R1, AE3F2 and AE3R2 were dissolved in sterile double distilled water to form a solution with a concentration of 10uM. The prepared primer solution was mixed according to the following ratio AE2R2:AE3R1:AE2F1:AE3F1:AE2R1:AE3F2:AE3R2=2:2:1:1:0.1:0.1:0.1 (V / V), and mixed evenly.
[0095] Prepare D Mix solution: Prepare 5 mM dNTP solution (containing 5 mM each of dATP, dTTP, dGTP and dCTP) with sterile double distilled water.
[0096] 2 ul of the DNA extracted from the specimens described in Example 1 were taken in PCR reaction tubes and placed in an ice bath. Add 2 ul of 10X buffer solution; 5 ...
Embodiment 3
[0098] Embodiment three, the PCR amplification of the second and third exon of HLA-B gene
[0099] Preparation of P Mix solution: primers BE2F1:BE2R1:BE2R2:BE3F1:BE3R1 were dissolved in sterile double distilled water to form a solution with a concentration of 10uM. The prepared primer solution was mixed according to the following ratio BE2F1:BE2R1:BE2R2:BE3F1:BE3R1=1:2:0.1:1.5:3 (V / V), and mixed evenly.
[0100] The DNA extracted from the specimen described in Example 1 was loaded and amplified by PCR according to the method of Example 2.
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Abstract
The invention involves biomedical field, particularly involves the detecting method and agent of the human leukocyte antigen genotyping. The method comprises the steps of: designing and synthesizing a set of probe by selecting the main variability region of three sites of HLA-A, -B, -DRB1 with most abundant polymorphism in human leukocyte antigen gene complexes, covalent-linking the probe to the fluorescent microspheres vector surface, each different fluorescent microspheres coupling a probe, balanced-mixing probes with different fluorescent microspheres to compose the liquid gene chip. The invention carries out balanced amplification of a number of gene fragments, the probe hybrids with target gene fragments in liquid environment, which has more hybridization efficiency compared with liquid-solid phase hybridization of traditional gene chip. In addition, the hybridization can be carried out in the ordinary 96-well plates, facilitates the pipetting operation with the use of multi-channel pipetting gun, the whole hybridization is convenient, rapid, which can effectively carry out high-throughput gene-typing.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a detection method and a reagent for human leukocyte antigen (Human Leukocyte Antigen, HLA) genotyping. Background technique [0002] The HLA gene is located on human chromosome 6p21.3, with a total length of about 4000kb, and is the most complex system of genetic polymorphism in human genes. HLA is the gene complex with the highest polymorphism among known genes so far, and there are about 2000 alleles known. According to the structure and distribution characteristics, the HLA complex can be divided into three types, which are called class I, II, and III genes respectively. Class I genes include ten kinds of HLA-A, -B, -C, -E, -F, -G, -H, -J, -K, -L. There are 27 types of class II genes, such as DR, DP, and DQ series, and their molecules are mainly distributed on the surface of antigen-presenting cells. In addition, there are some class III genes that make up the complement compone...
Claims
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