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HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof

An HLA-A and genotyping technology, applied in the field of nucleic acid sequencing and PCR sequencing, can solve the problems of DNA length not too long and read length short, and achieve the effect of good specificity and good primer conservation.

Active Publication Date: 2010-12-22
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the first-generation sequencing technology (sequencing technology based on the principle of Sanger sequencing), the length of DNA that can be used for sequencing library preparation of the new sequencing technology should not be too long (the maximum applicable length of Illunina GA is 700bp), plus The read length of the new sequencing technology is generally short, and the current Illumina GA bidirectional read length can only reach 200bp, and the PCR primers originally used for the HLA-SBT method are no longer applicable

Method used

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  • HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof
  • HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof
  • HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] sample extraction

[0044] DNA was extracted from 95 blood samples with known HLA-SBT typing results (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "China Bone Marrow Bank")) using a KingFisher automatic extractor (Thermo Company, USA). The main steps are as follows: Take out 6 deep-well plates and 1 shallow-well plate matched with the Kingfisher automatic extractor, add a certain amount of matching reagents according to the instructions and mark them, and place all the well-plates with reagents in the For the corresponding position, select the program "Bioeasy_200ul Blood DNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. After the program is finished, about 100ul of the eluted product collected in the plate Elution is the extracted DNA.

Embodiment 2

[0046] Make different PCR index primers by synthesizing PCR primers with different primer tags at the 5' end, so that different PCR index primers can be used for different samples, the PCR primers are 2, 3, 4 for HLA-A / B PCR primers for exon No. 1 and exon No. 12 of HLA-DRB12. Thereafter, primer tags are introduced at both ends of the PCR products by PCR reaction, thereby specifically labeling the PCR products from different samples.

[0047] 95 sets of PCR indexing primers were used to amplify 95 DNA samples respectively, each set of PCR indexing primers consisted of a pair of bidirectional primers (Table 2) and were used to amplify exons 2, 3, and 4 of HLA-A / B and The PCR primers (Table 3) of exon 12 of HLA-DRB consist of a pair of forward primer tags connected to the 5' end of each forward PCR primer, and a pair of forward primer tags connected to the 5' end of the reverse PCR primer. Reverse primer label to primer label. Primer tags are added directly to the 5' ends of P...

Embodiment 3

[0071] PCR product pooling and purification

[0072] Take 20ul from the remaining PCR products of HLA-P-A2 in the 96-well plate (except the negative control) and mix them in a 3ml EP tube, labeled as HLA-A2-Mix, and do the same for the other 6 96-well plates , respectively marked as HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix, shake and mix, from HLA-A2-Mix , HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix each take 200ul and mix in a 3ml EP tube, label For HLA-Mix, get 500ul of DNA mixture from HLA-Mix and pass it through the Qiagen DNA Purification kit (QIAGEN company) for column purification (see instructions for specific purification steps), and 200ul of purified DNA is measured by Nanodrop8000 (Thermo Fisher Scientific company) The concentration of HLA-Mix DNA is 48ng / ul.

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Abstract

The invention provides an HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer, and a method of applying the PCR primer to HLA gene analysis based on a sequencing method.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to the technical field of PCR sequencing. In particular, the present invention provides PCR primers for HLA-A, B (second generation sequencing) genotyping. On the other hand, the method of the present invention relates to the typing method of DNA sequence, especially the high-resolution typing method of HLA gene. Background technique [0002] Human leukocyte antigen, namely HLA (human leukocyte antigen, HLA), is one of the most polymorphic gene systems found so far. It is closely related to the rejection of allogeneic organ transplantation. The study found that, at the time of transplantation, the higher the degree of HLA-related gene matching between the donor and the recipient, the higher the resolution and the longer the survival time of the graft. [0003] The current international standard HLA genotyping techniques include PCR-SSP (sequence-specific primer po...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6881C12Q2600/16
Inventor 李剑刘莹陈仕平张彩芬
Owner BGI GENOMICS CO LTD
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