PCR primer set for HLA gene, and sequencing method using same

A technology of HLA-C and HLA-DRB1, which is applied in the field of efficient and highly uniform sequencing, can solve the problems of difficult to detect alleles, reduce the uniformity of amplification and collection efficiency, and prevent non-specific amplification and reduce HLA Type cost, effect of reducing wrong determination

Inactive Publication Date: 2019-11-22
KYOTO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, as many as 10,000 probes have been designed to cover the sequences of as many HLA alleles registered in the database as possible, which in turn causes problems of reduced amplification uniformity and collection efficiency
In addition, it is difficult to detect alleles other than the covered HLA alleles, and genomic fragments of genes other than the target gene can be mixed

Method used

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  • PCR primer set for HLA gene, and sequencing method using same
  • PCR primer set for HLA gene, and sequencing method using same
  • PCR primer set for HLA gene, and sequencing method using same

Examples

Experimental program
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Effect test

Embodiment 1

[0089] Embodiment 1 The design of the multiplex PCR primer set of classical HLA gene

[0090] A procedure for designing primers for multiplex PCR of the six classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) is described. Bowtie2 (version 4.1.2) was used to map all reads obtained by the next generation sequencer.

[0091] I. Assembly of full-length genes from sequencing results of HLA genes

[0092] 1. Sequencing of HLA genes by conventional methods

[0093] Using an existing primer set [Hosomichi, K. et al. (2013) Phase-defined complete sequencing of the HLA genes by next-generation sequencing. BMC Genomics, 14:355.], 768 samples (unreleased samples) were Multiplex PCR with 2 panels (3 genes per panel) (see Table 2). For DRB1, primers with modified sequences were added. For the experimental conditions, refer to Table 3. The obtained amplification products were sequenced by MiSeq.

[0094]

[0095]

[0096] 2. Mapping to HLA dictionary

[0097]For the ...

Embodiment 2

[0174] Example 2 Multiplex PCR and NGS sequencing

[0175] I. Long PCR

[0176] 1. A total of 384 DNA samples were each subjected to multiplex long PCR targeting 6 genes of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1. Amplifications were performed in the corresponding two pools below.

[0177] Set 1: HLA-A, -C, -DPB1(FF, FR), -DRB1(FF, FR)

[0178] Set 2: HLA-B, -DQB1, -DPB1(RF, RR), -DRB1(RF, RR)

[0179] Primer sequences and final concentrations are shown in Table 3. Prepare the following for PCR.

[0180] DNA 25ng

[0181] 0.25 μL of Tks Gflex TM DNA polymerase

[0182] 6.25 μL 2 × Gflex TM PCR buffer

[0183] After preparation to a final volume of 12.5 μL, apply Cool Start TM method (TaKaRa Inc.), and PCR reactions were performed under the temperature conditions and cycle numbers shown in Table 5. As a PCR device, use GeneAmp R PCR System 9700 (Thermo Fisher Scientific inc., Waltham, MA, USA).

[0184] 2. By Agencourt R AMPure R Amplified products were purifie...

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Abstract

The present invention provides a primer set for use in HLA gene amplification, which contains a primer comprising a nucleotide sequence represented by any one SEQ ID NOs: 1 to 16, and also provides ahigh-throughput high-uniform sequencing method using the primer set.

Description

[technical field] [0001] The present invention relates to a set of PCR primers for classical HLA genes, and an efficient and highly uniform sequencing method using the same. [Background technique] [0002] Human leukocyte antigen (HLA), which is the major histocompatibility complex (MHC) of human, is an important protein related to immune response and deeply involved in immune-related diseases. The gene region encoding HLA is located on the short arm of human chromosome 6, 6p21.3, and the important genes of HLA include HLA-A, HLA-B, HLA-C, which belong to class I molecules expressed in almost all cells, and belong to HLA-DRB1, HLA-DQB1, HLA-DPB1 of class II molecules expressed in cells in the system. These HLA genes are deeply involved in histocompatibility in organ transplantation, and accurate determination (typing) of HLA alleles is extremely important clinically. However, the above-mentioned 6 HLA genes are regions rich in polymorphism in the human genome, with more th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/6869
CPCC12N15/09C12Q1/68C12Q1/6881C12Q2535/122C12Q1/6869C12Q1/6844C12Q1/686
Inventor 松田文彦川口修治清水正和日笠幸一郎
Owner KYOTO UNIV
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