CN115354072A - Primer group for HLA-DQA1 genotyping and analysis method - Google Patents
Primer group for HLA-DQA1 genotyping and analysis method Download PDFInfo
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Abstract
The invention discloses a primer group and an analysis method for HLA-DQA1 genotyping, wherein the primer group comprises a mixture of two upstream primers and four downstream primers. The typing method comprises the steps of genome DNA extraction, gene amplification product purification, gene amplification product interruption, library construction, second-generation sequencing and genotyping. The invention can effectively improve the detection efficiency, improve the accuracy of genotyping, reduce the detection cost and simplify the operation steps, and can be widely applied to the field of HLA genotyping and detection.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer group and an analysis method for HLA-DQA1 genotyping.
Background
The human HLA-DQA1 gene encodes the alpha 1 subunit of HLA-DQ MHC cell surface receptor, and HLA-DQ molecules are heterodimers composed of an alpha chain (DQA) and a beta chain (DQB) immobilized on a membrane, which play a central role in the immune system by presenting protein polypeptides from outside the cell. HLA-DQA1 belongs to class II Human Leukocyte Antigen (HLA) genes, important genes of HLA include class I genes HLA-A, HLA-B, HLA-C expressed in almost all cells, and class II genes HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc. mainly expressed in immune system cells. HLA class I and II genes are located in the short arm 6p 21.31 region of chromosome 6, which is the region with the highest gene density and most abundant polymorphism in the known human chromosome. HLA genes are highly polymorphic, with many different alleles present, thus carefully regulating the acquired immune system. The human leukocyte antigen determines histocompatibility when transplantation is performed, and the more similar the human leukocyte antigens of the donor and recipient, the smaller the rejection response. In addition, specific forms of HLA proteins have been shown to be highly associated with, or determined to induce, a particular disease. HLA-DQA1 gene has abundant polymorphism, 383 HLA-DQA1 alleles recorded in IPD-IMGT/HLA database at present.
Some single nucleotide polymorphisms in the HLA-DQA1 gene have been shown to be associated with diseases such as HLA-DQA1 x 05 and DQA1 x 03 genotypes, which are associated with not only diabetes and rheumatoid arthritis, but also increased risk of celiac disease in carriers of those who have a chronic autoimmune intestinal disease caused by ingestion of gluten-containing foods, so that such people should avoid ingesting cereals such as wheat, barley, rye, etc. to reduce the risk of celiac disease. In addition, studies have shown that multiple HLA-DQA1 genotypes are associated with susceptibility to systemic lupus erythematosus. Therefore, the typing detection of the HLA-DQA1 gene has certain guiding significance for definite diagnosis and treatment direction of some diseases. However, there are few typing detection kits for human HLA-DQA1 gene currently, and CN109913539A discloses a method for typing 14 HLA genes including DQA1 gene based on targeted capture, but the probe has a certain binding force, and when the probe and the capture region contain more new variations, the capture efficiency may be greatly reduced.
The research on HLA typing techniques was first focused on serological and cytological studies, which focused on analyzing HLA product specificity, and had more limitations and less accuracy. With the development of gene sequencing technology, DNA sequence-based HLA typing methods have gradually replaced serological and cytological typing methods, including restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction oligonucleotide probe hybridization (PCR-SSO), sequence specific primer method (PCR-SSP), polymerase chain reaction single strand conformation polymorphism (PCR-SSCP), etc., which have the disadvantages of limited resolution and inability to identify new sites, although they have high sensitivity and low cost. The direct sequencing typing method (PCR-SBT) is the 'gold standard' of the HLA typing method recommended by the World Health Organization (WHO), and although the resolution is high, the result is easy to be uncertain, and the ambiguous result is easy to be caused. The current PCR scheme is combined with a second-generation sequencing technology to better solve the problems, sequences of HLA gene exon and intron regions are obtained through specific primer amplification and second-generation sequencing, and an HLA typing result with ultrahigh resolution can be obtained through analysis.
Disclosure of Invention
The present invention is directed to overcoming the above problems and providing a primer set and an analysis method for HLA-DQA1 genotyping. In order to achieve the purpose, the invention adopts the following technical scheme:
according to the invention, through analyzing 1463 whole genome sequencing data of Chinese people, the crowd frequency information of the upstream and downstream sequences of the HLA-DQA1 gene is obtained, primer design is carried out in the upstream and downstream intervals of the HLA-DQA1 based on the crowd frequency information, the primers are required to avoid the region containing more single nucleic acid polymorphisms (SNP) as far as possible, and meanwhile, a plurality of primer sequences are arranged in the region containing the single nucleic acid polymorphisms to contain polymorphic bases, so that the primers can better amplify the HLA-DQA1 gene of the Chinese people, meanwhile, the primer design is carried out in the upstream and downstream intervals of the HLA-DQA1 gene, the full-length sequence of the HLA-DQA1 can be amplified and sequenced, and further, an ultra-high-resolution HLA-DQA1 typing result can be obtained; based on the method and the principle, obtaining a primer for HLA-DQA1 genotyping, wherein an upstream primer set used for HLA-DQA1 genotyping is a mixture containing two primers, and a downstream primer set is a mixture containing four primers; the annealing temperature of the primer design is 65 ℃, the amplification length is 7037bp, and all exon and intron regions of the HLA-DQA1 gene are included; the sequences of the upstream and downstream primer groups are specifically as follows:
an upstream primer:
HLA-DQA1-1F：5’-AGGTAGGTGGGGTCAGCTTAACATTT-3’
HLA-DQA1-2F：5’-AGGTAGGTGGGGTCAACTTAACATTT-3’；
a downstream primer:
HLA-DQA1-1R：5’-TGGCTTCTGTGTTACTGGACATGAA-3’
HLA-DQA1-2R：5’-TGGCTTCTGTGTTACTGGATATGAC-3’
HLA-DQA1-3R：5’-TGGCTTCTGTGTTACTGGACATGAC-3’
HLA-DQA1-4R：5’-TGGCTTCTGTGTTACTGGATATGAA-3’。
the analysis method of the invention comprises the following steps:
s1, extracting genome DNA
Extracting the genome DNA of a sample to be detected by using a nucleic acid extraction kit, and determining the concentration and the purity;
s2. Gene amplification
Configuring an amplification reaction system by using specific primers and other related reagents, and carrying out PCR amplification on a target region of a gene according to a certain amplification procedure;
s3, purifying gene amplification products
Purifying the amplified target gene fragment by adopting a DNA product purification kit to ensure the concentration and the purity of the fragment;
s4, gene amplification product interruption
Breaking the obtained DNA fragments by using an ultrasonic breaking method to uniformly fragment the DNA;
s5. Library construction
Performing end modification and library enrichment on the fragmented DNA by using a library building kit; the prepared DNA library is subjected to length distribution and concentration detection by using a bioanalyzer;
s6. Second generation sequencing
Performing second-generation sequencing on the prepared library;
s7, genotyping
And (3) carrying out HLA genotyping analysis on the sequenced sequence to determine the genotype of HLA-DQA 1.
As an improvement, the reaction system in step S2 is: 2xPCR Master Mix 12.5. Mu.L, dNTP 0.5. Mu.L, phanta Max Super-Fidelity DNA Polymer 0.5. Mu.L, forward primer 0.5. Mu.L, reverse primer 0.5. Mu.L, template DNA 1. Mu.L, ddH 2 O 9.5μL。
As a modification, the PCR amplification procedure in step S2 is:
as an improvement, the fragment length of the product after being broken in the step S4 is distributed between 100-400bp, and the main peak is 180-220bp.
The invention has the advantages that:
the invention can effectively improve the detection efficiency, improve the accuracy of genotyping, reduce the detection cost and simplify the operation steps, and can be widely applied to the field of HLA genotyping and detection.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of 6 samples of HLA-DQA1 gene.
FIG. 2 is a quality control chart of HLA-DQA1 gene after disruption.
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples so as to facilitate the understanding of the present invention, but the following examples do not limit the scope of the present invention.
Example 1
The embodiment discloses a primer group and an analysis method for HLA-DQA1 genotyping, wherein the primer comprises the following components:
an upstream primer:
HLA-DQA1-1F：5’-AGGTAGGTGGGGTCAGCTTAACATTT-3’
HLA-DQA1-2F：5’-AGGTAGGTGGGGTCAACTTAACATTT-3’；
a downstream primer:
HLA-DQA1-1R：5’-TGGCTTCTGTGTTACTGGACATGAA-3’
HLA-DQA1-2R：5’-TGGCTTCTGTGTTACTGGATATGAC-3’
HLA-DQA1-3R：5’-TGGCTTCTGTGTTACTGGACATGAC-3’
HLA-DQA1-4R：5’-TGGCTTCTGTGTTACTGGATATGAA-3’。
the analysis method using the above primer set comprises the steps of:
s1, extracting genome DNA
Extracting genome DNA of 6 samples to be tested by using a nucleic acid extraction kit, and measuring the concentration and the purity by using Nanodrop2000, wherein the DNA concentration is 40-200 ng/mu L, and the OD is 260/280 The ratio should be between 1.7 and 2.0, and the DNA concentration is diluted to 40 ng/. Mu.L for use.
S2 HLA-DQA1 gene amplification
Preparing a reaction system shown in a table 1 by using a specific primer group and other related reagents, and performing PCR amplification on a DNA target region according to a PCR amplification program shown in a table 2; after the PCR amplification reaction is finished, 5 mu L of amplification product is subjected to agarose gel electrophoresis to confirm the amplified target fragment, as shown in figure 1, the amplified fragment has correct size and clear target band, the stability of PCR amplification is proved, and the amplification product is single and has no non-specific amplification.
TABLE 1 PCR reaction System
TABLE 2 PCR amplification procedure
Example 2
The analysis method of the embodiment is as follows:
s1. Extraction of genomic DNA
Genomic DNA of 8 clinical samples was extracted using a nucleic acid extraction kit, and the concentration and purity of the extracted genomic DNA were measured using a Nanodrop2000, where the DNA concentration was 40-200 ng/. Mu.L and OD 260/280 The ratio should be between 1.7 and 2.0, the DNA concentrationDiluted to 40 ng/. Mu.L for use.
S2 HLA-DQA1 gene amplification
According to the specific primer group in the example 1 and other related reagents, preparing the reaction system shown in the table 1, and carrying out PCR amplification on the target region of the DNA according to the PCR amplification procedure shown in the table 2; after the PCR amplification reaction was completed, 5. Mu.L of the amplified product was subjected to agarose gel electrophoresis to confirm the amplified target fragment.
S3, purification of HLA-DQA1 gene amplification product
Purifying the amplified target gene fragment according to the operation instruction of the DNA product purification kit, measuring the concentration and purity of the purified product by using Nanodrop2000, and determining OD 260/280 The ratio should be 1.7-1.9.
S4 disruption of the product after HLA-DQA1 Gene purification
Breaking the purified product by using an ultrasonic breaking instrument to enable DNA to be fragmented uniformly, carrying out concentration measurement on the broken product by using the Qubit 4.0, and carrying out fragment length measurement by using an Agilent bioanalyzer, wherein the fragment length is mainly distributed between 100 and 400bp, and the main peak is about 200bp, as shown in figure 2.
S5. Library construction
According to the operation instruction of the library building kit, performing terminal modification, adaptor connection and library amplification reaction on the fragmented DNA, wherein the amplification reaction program is as follows: pre-denaturation at 98 ℃ for 45s; denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and 6 cycles are performed; extending for 1min at 72 ℃;4 ℃ and infinity. The prepared DNA library is subjected to concentration measurement by using the Qubit 4.0 and fragment length measurement by using an Agilent bioanalyzer, so that the fragment length is mainly distributed between 200 and 500bp, and the main peak is about 300 bp.
S6. Second generation sequencing
And (3) performing on-machine sequencing on the library with qualified quality control, performing sequencing by using a HiSeq 4000 sequencer of Illumina, and performing sequencing with the sequencing depth of 1000 x by adopting a sequencing mode with 150bp at both ends.
S7 HLA-DQA1 genotyping
The data after the sequencing is off-machine is compared and analyzed with an IMGT/HLA database (version 3.43) through HLAscan software to obtain the typing information of the HLA-DQA1 gene, the genotypes of 8 clinical samples are determined, the obtained typing results are respectively compared with the typing results obtained by Whole Exome Sequencing (WES), and the results in the table 3 show that the experimental typing method is completely consistent with the typing results obtained by the whole exome sequencing, thereby illustrating the accuracy and high resolution of the genotyping method.
TABLE 3 comparison of HLA-DQA1 genotyping results for consistency
The embodiments of the present invention have been described in detail above, but they are merely exemplary, and the present invention is not equivalent to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered in the present invention.
Claims (5)
1. A primer set for HLA-DQA1 genotyping, wherein the upstream primer set for HLA-DQA1 genotyping comprises a mixture of two primers, and the downstream primer set comprises a mixture of four primers; the sequences of the upstream primer group and the downstream primer group are specifically as follows:
an upstream primer:
HLA-DQA1-1F：5’-AGGTAGGTGGGGTCAGCTTAACATTT-3’
HLA-DQA1-2F：5’-AGGTAGGTGGGGTCAACTTAACATTT-3’；
a downstream primer:
HLA-DQA1-1R：5’-TGGCTTCTGTGTTACTGGACATGAA-3’
HLA-DQA1-2R：5’-TGGCTTCTGTGTTACTGGATATGAC-3’
HLA-DQA1-3R：5’-TGGCTTCTGTGTTACTGGACATGAC-3’
HLA-DQA1-4R：5’-TGGCTTCTGTGTTACTGGATATGAA-3’。
2. an analysis method using the primer set for HLA-DQA1 genotyping according to claim 1, comprising the steps of:
s1, extracting genome DNA
Extracting the genome DNA of a sample to be detected by using a nucleic acid extraction kit, and determining the concentration and the purity;
s2. Gene amplification
Configuring an amplification reaction system by using a specific primer and other related reagents, and carrying out PCR amplification on a target region of the gene according to a certain amplification procedure;
s3, purifying gene amplification products
Purifying the amplified target gene fragment by adopting a DNA product purification kit to ensure the concentration and purity of the fragment;
s4. Disruption of gene amplification products
Breaking the obtained DNA fragments by using an ultrasonic breaking method to uniformly fragment the DNA;
s5. Library construction
Performing end modification and library enrichment on the fragmented DNA by using a library building kit; the prepared DNA library is subjected to length distribution and concentration detection by using a bioanalyzer;
s6. Second generation sequencing
Performing second-generation sequencing on the prepared library;
s7, genotyping
And comparing the sequence obtained by sequencing with an HLA gene database to determine the genotype of the HLA-DQA 1.
3. The method for analyzing primer set for HLA-DQA1 genotyping according to claim 2, wherein the reaction system in step S2 is: 2xPCR Master Mix 12.5. Mu.L, dNTP 0.5. Mu.L, phanta Max Super-Fidelity DNA Polymer 0.5. Mu.L, forward primer 0.5. Mu.L, downstream primer 0.5. Mu.L, template DNA 1. Mu.L, ddH 2 O9.5μL。
4. The method for analyzing primer set for HLA-DQA1 genotyping according to claim 2, wherein the PCR amplification procedure in step S2 is:
5. the method for analyzing primer set for HLA-DQA1 genotyping according to claim 2, wherein the length of the cleaved fragments of the product in step S4 is between 100-400bp, and the main peak is 180-220bp.
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