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Hla gene multiplex dna typing method and kit

A technology of DNA typing and HLA-A, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems that the PCR conditions are not completely unified, and the PCR multiplex method has not yet been achieved. The effect of speed and simplicity

Active Publication Date: 2016-02-17
GENODIVE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, the PCR conditions used for each gene are not completely unified, and a multiplex method that simultaneously performs PCR for all loci has not yet been achieved.
[0014] Furthermore, there is no study on a high-speed PCR device that greatly shortens the time required for temperature rise or fall when performing PCR in which rapidity of PCR is expected.

Method used

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  • Hla gene multiplex dna typing method and kit
  • Hla gene multiplex dna typing method and kit
  • Hla gene multiplex dna typing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0161] [Purpose]

[0162] The amplification status of each HLA gene was checked using PrimeSTAR GXL DNA Polymerase (TaKaRa) as an enzyme.

[0163] [method]

[0164] PCR was performed using PrimeSTAR GXL DNA Polymerase (TaKaRa), using the extracted genomic DNA as a template, and each gene-specific primer set of HLA class I and HLA class II (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.

[0165] (1) Add 4 μL of 5xPrimeSTAR GXL buffer solution, 1.6 μL of dNTP solution, 1-3 μL (4 pmol / μL) of PCR primers, and 0.4 μL of Prime STAR GXL polymerase to 50 ng of genomic DNA solution. The total volume was adjusted to 20 μL with sterile water.

[0166] (2) After keeping this at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 70° C. for 5 minutes were repeated 30 times as one process. In this PCR, GeneAmp PCR System 9700 (Life Technologies) was used. After PCR, the amplification status of PCR products was confirmed by agarose gel elec...

Embodiment 2）

[0170] [Purpose]

[0171] The purpose is to confirm the amplification status of each HLA gene using Tks Gflex DNA Polymerase (TaKaRa) as an enzyme.

[0172] [method]

[0173] Using Tks Gflex DNA Polymerase (TaKaRa), the extracted genomic DNA was used as a template, and PCR was performed using HLA class I and HLA class II gene-specific primer sets (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.

[0174](1) Add 10 μL of 2xGflx PCR Buffer, 1-3 μL (4 pmol / μL) of PCR primers, and 0.2 μL of Tks Gflex DNA Polymerase to 50 ng of genomic DNA solution, and adjust the total amount of the reaction solution with sterilized water 20 μL.

[0175] (2) After heat-retaining at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 68° C. for 5 minutes were repeated 10 times as one process. Then, two steps of 10 seconds at 98° C. and 5 minutes at 65° C. were repeated 20 times as one process. In this PCR, GeneAmp PCR System 9700 (Life Technologies)...

Embodiment 3）

[0180] [Purpose]

[0181] Confirm the difference in PCR status between the general-purpose PCR equipment, GeneAmp PCR System 9700 (Life Technologies) and the high-speed amplification equipment specifically used for PCR, PCR Thermal Cycler Fast (TaKaRa), and study the time required for PCR .

[0182] [method]

[0183] Using Tks Gflex DNA Polymerase (TaKaRa), the extracted genomic DNA was used as a template, and PCR was performed using HLA class I and HLA class II gene-specific primer sets (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.

[0184] (1) Add 10 μL of 2xGflex PCR Buffer, 1-3 μL (4 pmol / μL) of PCR primers, and 0.2 μL of Tks Gflx DNA Polymerase to 50 ng of genomic DNA solution, and adjust the total amount of the reaction solution to 20 μL.

[0185] (2) After heat-retaining at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 68° C. for 5 minutes were repeated 10 times as one process. Then, two steps of 10 seconds at ...

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Abstract

The purpose is to provide a high-precision DNA typing method and kit that make full use of a high-throughput sequencer and eliminate the ambiguity derived from phase ambiguity. The invention provides an HLA DNA typing method characterized by including: (1) a step for providing a set of primers that each hybridize specifically to an upstream region and a downstream region of at least two genes selected from genes belonging to HLA class I and class II in the nucleotide sequence of the human genome and amplify under identical PCR conditions; (2) a step for amplifying these at least two genes in a test sample (DNA) simultaneously in the same container under identical PCR conditions using this set of primers; (3) a step for determining the nucleotide sequence of the PCR product; and (4) an optional step for conducting a homology search of a database.

Description

technical field [0001] The invention relates to an ultrahigh-resolution multiplex DNA typing method for HLA genes using a high-throughput DNA sequencer. Background technique [0002] Human Leukocyte Antigen (HLA), which is the major histocompatibility complex (MHC) of humans, is closely involved in immunity by presenting peptides from foreign proteins such as pathogens and peptides from self-proteins to T cells Induction of response, six antigens are known as the main HLA. That is, class I antigens (HLA-A, HLA-B, HLA-C) expressed in almost all cells and class II antigens (HLA-DR, HLA-DQ, HLA-DP) mainly expressed 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 antigen is encoded by the genes of HLA-A, HLA-B, and HLA-C, and the β chain of class II ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q2600/156C12Q2600/16C12Q1/6881
Inventor 椎名隆铃木进悟和田有纪光永滋树猪子英俊
Owner GENODIVE PHARMA
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