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dna typing method and kit for hla gene

A technology of HLA-A and HLA-DRB1, which is applied in the field of DNA typing of HLA genes and kits, can solve problems such as difficult and high-precision HLA typing, inaccurate determination of cis-trans positional relationship, phase ambiguity, etc., and achieve high The effect of precision HLA matching

Active Publication Date: 2016-08-17
GENODIVE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] These conventional DNA typing methods have the advantage of being able to quickly type most samples, but sometimes they cannot accurately determine the cis-trans position relationship of exons on the chromosome in the case of polymorphic regions and class I genes, so phase ambiguity occurs (phase ambiguity), sometimes it is difficult to perform high-precision HLA typing
[0011] In addition, the conventional method is a DNA typing method based on the application of PCR centering on the exon region of each gene. Therefore, the base substitution in the intron region and the promoter region is ignored. As a result, the gene structure and other expression Detection of null alleles with identical HLA genes but suppressed expression may fail

Method used

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  • dna typing method and kit for hla gene
  • dna typing method and kit for hla gene
  • dna typing method and kit for hla gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0192] [experimental method]

[0193] 1. Using the extracted genomic DNA as a template, a PCR reaction was performed using specific primer sets for each HLA class I gene (refer to Table 1: sequence numbers 1 to 8). The specific sequence is as follows.

[0194] (1) Prime STAR GXL polymerase (TaKaRa) was used for PCR amplification. That is, in 50 ng of genomic DNA solution, add 4 μL of 5 × PrimeSTAR GXL buffer, 1.6 μL of dNTP solution, 4 μL (1 pmol / μL) of PCR primers, 0.8 μL of Prime STAR GXL polymerase, and inactivate Bacterial water to adjust the total amount of the reaction solution to 20 μL.

[0195] (2) After keeping warm at 94°C for 2 minutes, then repeat 30 times as a single process of 3 steps of 98°C for 10 seconds, 60°C for 20 seconds, and 68°C for 5 minutes. In addition, GeneAmp PCR System 9700 (Applied Biosystems) was used for this PCR amplification. After PCR, the amplification of PCR products was confirmed by agarose gel electrophoresis. Its electrophoresis is...

Embodiment 2）

[0205] [experimental method]

[0206] 1. Using the extracted genomic DNA as a template, PCR reactions were performed using specific primer sets for HLA class I and HLA class II genes (refer to Tables 1 to 4: sequence numbers 1 to 8, 9 to 22, and 31 to 50). The specific sequence is as follows.

[0207] (1) Prime STAR GXL polymerase (TaKaRa) was used for PCR amplification. That is, to 50 ng of genomic DNA solution, add 4 μL of 5 × PrimeSTAR GXL buffer, 1.6 μL of dNTP solution, 1 to 7 μL (4 pmol / μL) of PCR primers, 0.8 μL of Prime STAR GXL polymerase , and adjust the total amount of the reaction solution to 20 μL with sterilized water.

[0208] (2) After keeping warm at 94°C for 2 minutes, then repeat 2 steps of 98°C for 10 seconds and 70°C for 5 minutes as one process 30 times. In addition, GeneAmp PCR System 9700 (Applied Biosystems) was used for this PCR amplification. After PCR, the amplification of PCR products was confirmed by agarose gel electrophoresis. Its electrop...

Embodiment 3）

[0218] [experimental method]

[0219] 1. Genomic DNA was extracted using Buccal Cell DNA Extraction Kit, BuccalQuick (TRIMGEN).

[0220] 2. Genomic DNA extracted using Buccal Cell DNA Extraction Kit, BuccalQuick (TRIMGEN) was further purified by isopropanol and ethanol.

[0221] 3. Genomic DNA was extracted using QIAamp DNA Blood Mini Kit (QIAGEN).

[0222] 4. Apply each of the 3 samples of genomic DNA extracted in items 1 to 3, and use the same experimental method as in Example 1 and Example 2 to apply HLA-A, HLA-B, HLA-C, and HLA-DQB1 specific primer sets (Refer to Table 1 and Table 4: Sequence Nos. 1 to 8, 29, 30, 48 to 50) PCR reactions were carried out. After PCR, the amplification of PCR products was confirmed by agarose gel electrophoresis. Its electrophoresis is shown in Figure 7.

[0223] [Experimental Results and Discussion]

[0224] Figure 7 Swimming lanes 1 to 3 show the amplification of PCR products under the condition of extraction with experimental ...

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Abstract

The purpose of the present invention is to provide a high-precision DNA typing method and kit, which eliminates the ambiguity caused by phase ambiguity. The invention provides a DNA typing method for HLA, which is characterized by comprising the following steps: (1) preparing HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA ‑DPA1 and HLA‑DPB1 gene upstream region and downstream region specific annealing primer set and primer set specific annealing to exon 2 and 3' side untranslated region of HLA‑DRB1; (2) A step of performing PCR amplification of the test sample (DNA) using the aforementioned primer set; (3) a step of determining the base sequence of the PCR amplification product; and (4) optionally, performing a homology search with a database step.

Description

technical field [0001] The invention relates to a method and a kit for using a large-scale parallel sequence analyzer to carry out DNA typing of HLA genes. Background technique [0002] Human leukocyte antigen (HLA), which is the human major histocompatibility complex (MHC), is profoundly involved in the induction of immune responses by presenting to T cells peptides derived from foreign proteins such as pathogens as well as peptides derived from their own proteins, with 6 This antigen is known as the major HLA. That is, class I molecules (HLA-A, HLA-B, HLA-C) expressed in almost all cells and class II molecules (HLA-DR, HLA-DQ, HLA-DP) expressed mainly in cells of the immune system . [0003] HLA class I antigens are composed of highly polymorphic α chains and almost no polymorphic β2-microglobulins, and HLA class II antigens are composed of highly polymorphic β chains and low polymorphic α chains. The α chain of class I molecules is encoded by HLA-A, HLA-B, and HLA-C ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6881C12Q2600/156C12Q1/6888C12Q2600/16
Inventor 椎名隆铃木进悟尾崎有纪光永滋树猪子英俊
Owner GENODIVE PHARMA
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