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Multiple dna typing method and kit for hla gene

A DNA typing, HLA-A technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, biochemical equipment and methods, etc., can solve the problems that the PCR multiplex method has not yet been achieved, and the PCR conditions have not been completely unified.

Active Publication Date: 2021-10-08
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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  • Multiple dna typing method and kit for hla gene
  • Multiple dna typing method and kit for hla gene
  • Multiple dna typing method and kit for hla gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0161] [Purpose]

[0162] The amplification status of each HLA gene was confirmed 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 for 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 e...

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), PCR was performed using the extracted genomic DNA as a template, and each gene-specific primer set for 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.

[0174](1) Add 10 μL of 2x Gflx 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] It is necessary to check the difference in PCR status between the general-purpose PCR equipment, GeneAmp PCR System 9700 (Life Technologies) and the high-speed amplification equipment specifically for PCR, PCR Thermal Cycler Fast (TaKaRa). time.

[0182] [method]

[0183] Using Tks Gflex DNA Polymerase (TaKaRa), PCR was performed using the extracted genomic DNA as a template, and each gene-specific primer set for 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.

[0184] (1) Add 10 μL of 2x Gflex 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 with sterilized water 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 9...

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Abstract

The purpose of the invention is to provide a high-precision DNA typing method and kit that eliminates the ambiguity caused by phase ambiguity by using a high-throughput sequencer. The present invention provides a DNA typing method for HLA, which is characterized in that it comprises: (1) preparing at least two genes that are specifically related to at least two genes selected from HLA class I and class II genes in the base sequence of the human genome. The step of hybridizing the upstream region and the downstream region of the gene and amplifying the primer set under the same PCR conditions; (2) using the aforementioned primer set to simultaneously amplify the test sample in the same container under the same PCR conditions ( (3) the step of determining the base sequence of the PCR product; and (4) optionally, the step of performing a homology search with 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 (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 th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
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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