TW201300528A - Method for hla-dqb1 genotyping and related primers thereof - Google Patents

Abstract

A method for amplifying exons 2 and / or 3 of HLA-DQB1 gene and the specific primers used in the method are provided. And a method for genotyping HLA-DQB1 gene and the amplification primer pairs used in the method are also provided.

Description

HLA-DQB1 genotyping method and related primers

The present invention relates to the field of molecular biology, and in particular, to a method for genotyping HLA-DQB1, and specific primers for use in the method.

Human leukocyte antigen (HLA), one of the most highly recognized gene systems discovered to date, is the main gene system that regulates the human-specific immune response and determines individual differences in disease susceptibility. The rejection of allogeneic organ transplantation is closely related. According to the characteristics of HLA gene structure and distribution, it is divided into three types of molecules: HLA-I, HLA-II and HLA-III. Among them, HLA-I and HLA-II molecular functions are mainly related to immune rejection, HLA-III Molecular function is mainly related to the synthesis of immune-related partial complement systems and inflammation-related factors. The study found that the higher the degree of HLA-related gene matching between donor and recipient, the higher the resolution, the longer the graft survival time.

HLA-DQB1 belongs to the class of HLA-II, and its gene is about 6800 bp in length and consists of 5 introns and 6 exons. In 1996, it was found that the compatibility of HLA-DQB1 genotypes between donors and recipients had an effect on the survival rate of grafts. In recent years, studies have found DQB1 locus and dilated cardiomyopathy, viral hepatitis B, diabetes, and silver shavings. The incidence and prognosis of various diseases such as diseases are related.

The current international standard HLA typing technology includes PCR-SSP (PCR-Sequence Specific Primer), PCR-SSO (PCR-Sequence Specific Oligonucleotide, polymerase chain reaction-sequence-specific oligo Glycosylate) and PCR-SBT (PCR-Sequencing Based Typing, polymerase chain reaction - sequencing based typing).

The principle of HLA-SSP is to design a set of allele-specific primers, and obtain HLA-type specific amplification products by PCR technology, and determine the HLA type by electrophoresis analysis. The principle of HLA-SSO is to design an HLA type-specific oligonucleotide sequence as a probe, label the PCR product, and hybridize with the PCR product (gene DNA to be detected) and the probe. The HLA type is judged by detecting the fluorescent signal. The detection signals of HLA-SSP and HLA-SSO are analog signals, and the resolution can only reach the low level and can not detect the new allele.

The HLA-SBT (Sequence-based typing) technique is a high-resolution typing method for determining HLA genotypes by sequencing HLA-related gene sequences, which is characterized by intuitive, high-resolution and detection of new alleles. However, the existing HLA-SBT adopts the Sanger legal sequence (capillary micro-electrophoresis), and the complicated experimental procedure, low throughput and high experimental cost make it difficult to apply to large-scale HLA high-resolution typing projects.

HLA-SBT based on the second-generation sequencing technology represented by Illumina Solexa and Roche 454 (hereinafter referred to as the new sequencing technology) is also a method for determining the HLA genotype by directly measuring the nucleic acid sequence of the PCR-amplified DNA product. The high-resolution typing method, in addition to the original intuitive, high-resolution and detection of new alleles, has the characteristics of single-molecule sequencing, simple experimental procedure, high throughput and low cost. However, compared to the first-generation sequencing technique (sequencing technology based on the Sanger principle principle), the DNA length that can be used for the sequencing of the new sequencing technology gene bank cannot be too long (the current maximum length of Illumina Solexa) It is 700 bp), and the new sequencing technology is generally short. The current Illumia GA bidirectional read length can reach 300 bp.

In view of the characteristics of the new sequencing technology, the length of the PCR product should not exceed 700 bp, and the PCR primer of HLA-SBT based on the Sanger legal method is no longer applicable. Therefore, it is necessary to design a set of primers with good conservation and specificity and the length of the PCR product to meet the requirements of the new sequencing technology.

In order to use the second generation sequencing technology for HLA-DQB1 genotyping, the present invention provides PCR primers for amplifying exon 2 and/or exon 3 of HLA-DQB1, which are the sequence identifications shown in Table 1. Number: 1-4. These PCR primers have good conservation and specificity, and can cover the full-length sequences of exons 2 and 3 of HLA-DQB1 locus. The length of PCR products are less than 700 bp, which meets the requirements of normal Illumina Solexa sequencing. In addition, the primer of the present invention is also applicable to the Sanger legal order.

The present invention also provides a novel HLA-DQB1 exon 2 and/or exon 3 amplification method, characterized in that PCR amplification is performed using the amplification primer pair of the present invention, and the sequences of the amplification primer pairs are shown in Table 1.

Since the exon 2 and/or exon 3 of HLA-DQB1 can be amplified by a PCR reaction, the method of the present invention is particularly advantageously useful for HLA-DQB1 genotyping. Compared with the existing HLA-DQB1 genotyping method, since the method using the method of the present invention and the product of the amplification primer are controlled between 300 and 400 bp, the sequencing based on Illumina Solexa can be utilized for further typing. Technical HLA-SBT.

In a third aspect of the invention, there is provided a method of sequencing exons 2 and/or exon 3 of HLA-DQB1 in a sample comprising the steps of:

(1) providing a sample and extracting the DNA of the sample;

(2) using the PCR primer in Table 1 to amplify the DNA to obtain a PCR product, preferably purifying the PCR product;

(3) The PCR product is sequenced, and the sequencing method may be a second generation sequencing method such as Illumina Solexa or Roche 454.

In a fourth aspect of the invention, the invention provides an improved HLA-DQB1 genotyping method comprising:

(1) amplifying exon 2 and/or exon 3 of HLA-DQB1 to be tested using the PCR amplification primer pair of the present invention;

(2) The amplified exons are sequenced, and the sequencing results are compared with the standard sequences in the database to determine the genotyping results. The sequencing method may be a Sanger sequencing method, or may be a second generation sequencing method such as Illumina Solexa or Roche 454.

In another aspect, the present invention also provides a kit for performing HLA-DQB1 genotyping, which comprises a PCR amplification primer pair of the present invention. In one embodiment, the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.

The amplification primer pair and genotyping method provided by the present invention can be used for genotyping based on the amplification of exon 2 and/or exon 3 of HLA-DQB1. Compared to the prior art, the typing method utilizes Illumina Solexa sequencing technology, which has the characteristics of obtaining high-resolution HLA typing results with high throughput and low cost.

Detailed description of the preferred embodiment

Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art should understand that the following examples are merely illustrative of the invention and are not to be construed as limiting the scope of the invention.

The present invention adopts the following method to design a PCR primer pair for amplifying the exon 2 and/or exon 3 of HLA-DQB1, downloading all the latest HLA-DQB1 gene sequences from the IMGT/HLA internet site, and then saving them to a local disk. The HLA-DQB1 database was also downloaded; all the latest non-HLA-DQB1 HLA-II gene sequences were downloaded as a comparison database. The two databases were compared, and the conserved and specific sequences of each locus were searched for both ends and inside of exons 2 and 3, and the designed PCR primer sequences were compared with the human genome sequence for homology. When designing the PCR primer, try to ensure that the 3' end of the primer is specific, and ensure that the primer expands the specificity of the HLA-DQB1 gene. At the same time, the length of the PCR product is less than 700 bp, and the bonding temperature of the positive and negative primers is basically the same.

Multiple pairs of candidate HLA-DQB1 primers that meet the design requirements were used to amplify a small number of template DNAs with common serotypes of HLA-DQB1, and the most conservative and specific ones were screened for amplification of exons 2 and 3. 2 pairs of PCR primers for HLA-DQB1.

The DNA amplification method, the method of extracting DNA from a sample, the DNA purification method, and the DNA sequence alignment method involved in the present invention may be any available methods in the art. These methods can be selected by a person skilled in the art in the art, depending on the circumstances. For methods of DNA sequencing, those skilled in the art can perform the methods according to conventional methods or according to the instructions for use of the sequencing instrument.

For example, in the sequencing process using the second-generation sequencing technique, a label primer with a primer index sequence added at the 5' end can be used to interrupt the amplified PCR product and interrupt the product. End repair was performed and deoxyadenosine (A) was ligated at its 3' end, followed by a different PCR-free adaptor.

A sequence of tags is attached to the front end of the amplification primer to achieve simultaneous sequencing of multiple samples. Specifically, a primer primer can be synthesized by adding a primer index sequence at the 5' end of the PCR primer in combination with the PCR-index/barcode technique, and a unique primer tag is introduced for each sample in the PCR process. In this way, in the detection process using the second generation DNA sequencing technology, in addition to the PCR step must be processed one by one, other experimental links can be mixed together and processed simultaneously, and finally the detection result of each sample can pass its unique primer. The tag sequence is retrieved. The design of the primer label varies according to the experimental platform applied. Considering the characteristics of the Illumina GA sequencing platform itself, the present invention mainly considers the following points when designing the primer label: 1: Avoid more than 3 in the sequence of the primer label (including 3) single base repeats, 2: the total content of base A and base C in the same locus of all primer tags is between 30% and 70% of the total base content, 3: the primer tag sequence itself The GC content is between 40-60%, the sequence difference between 4: primer tags is greater than 4 bases, 5: the sequences with high similarity to the Illumina GA sequencing primer are avoided in the primer tag sequence, and the primer label is reduced. After the sequence was added to the PCR primer, the hairpin and dimer conditions caused by the PCR primer were observed.

The term "PCR-Free gene bank adapter" refers to a designed set of bases whose primary function is to assist in the immobilization of DNA molecules on a sequencing wafer and to provide a binding site for a universal sequencing primer, PCR-Free The gene bank adapter can be directly ligated to the DNA fragment in the sequencing gene pool by DNA ligase. The introduction process of the adapter is called PCR-Free gene library adapter because there is no PCR involved. "adapter" or "library adapter" tagging technique refers to the addition of different gene bank adapters to multiple sequencing gene banks [different gene pool adapters have different composition sequences The different parts of the sequence are called adapter indices, and the tag sequencing gene pool is constructed, so that multiple different tag sequencing gene pools can be mixed and sequenced, and finally the sequencing of each tag sequencing gene bank is completed. The result is a gene library tagging technique that can be distinguished from each other. For example, a PCR-FREE gene bank adapter is used in the embodiment of the invention from ILLUMIA.

In the following examples, 94 pairs of known HLA common genotypes were subjected to HLA-DQB1 locus PCR amplification using the selected 2 pairs of PCR primers, and the amplified products were subjected to Sanger method and second generation sequencing. The method is sequenced. The sequencing results were used for HLA-DQB1 typing and the conservatism and specificity of the PCR primers were verified by comparison with the original typing results.

Example 1 HLA-DQB1 genotyping using the second generation sequencing technique (Illumina Solexa) Sample extraction

DNA was extracted from a blood sample of the 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 Corporation, USA). The main steps are as follows: Take out the deep hole plate and one shallow hole plate of the six Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them. Place all the well plates with the reagents as required. In the corresponding position, select the program "Bioeasy_200ul Blood DNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. At the end of the program, 100 ul of eluted product in the plate Elution was collected as the extracted DNA.

2. PCR amplification

Different PCR tag primers can be made by synthesizing PCR primers with different primer tags at the 5' end, so that different PCR tag primers can be used for different samples. These PCR primers are for exons 2 and 3 of HLA-DQB1. PCR amplification primers. A primer tag is then introduced at both ends of the PCR product by a PCR reaction to specifically label PCR products from different samples.

94 sets of DNA primers were amplified by 94 sets of PCR primers. Each set of PCR primers was amplified by PCR primers for exon 2 or 3 of HLA-DQB1 (Table 1) and a pair of bidirectional primers. 2) Composition, wherein a forward primer tag of a pair of primer tags is connected to the 5' end of each forward PCR primer, and a reverse primer tag of a pair of primer tags is connected to the 5' end of the reverse PCR primer. The primer tag was directly added to the 5' end of the PCR primer when the primer was synthesized, and the primer was synthesized by Shanghai Invitrogen.

The 94 DNAs obtained in the sample extraction step were sequentially numbered 1-94, and the PCR reaction was carried out in a 96-well plate, and the DQB1 2 and 3 exons of each sample were amplified in the same reaction well. Two negative controls without a template were placed in the plate, and the primers used for the negative control corresponded to the PI-95 and PI-96. At the same time of the experiment, record the sample number information corresponding to each pair of primer labels.

The PCR procedure for HLA-DQB1 is as follows:

96 ° C 2 minutes

95°C 30 seconds → 60°C 30 seconds → 72°C 20 seconds (32 cycles)

15 ° C standing

The PCR reaction system of HLA-DQB1 is as follows:

Wherein PInf-Q-F2/3 indicates the F-introduction of HLA-DQB1 with the nth forward primer tag sequence (Table 1) at the 5' end of the primer, and PInf-QR 2/3 indicates that the 5' end of the primer has the nth The R primer of the HLA-DQB1 of the reverse primer tag sequence (here n≦96), and so on. And each sample corresponds to a specific set of PCR primers.

The PCR reaction was run on a Bio-Rad PTC-200 PCR machine. After the PCR was completed, 2 ul of the PCR product was detected by 1.5% agarose gel electrophoresis. Figure 1 shows the results of electrophoresis of 94 samples of HLA-DQB1 2+3 exon PCR products. The DNA standard molecular weight reference (M) is DL 2000 (Takara).

3. PCR product mixing and purification

From the remaining PCR product of the 96-well plate HLA-P-DQB1 (except the negative control), 20 ul each was mixed in a 3 ml EP tube, labeled HLA-Q-Mix and mixed well. 500 ul of DNA mixture from HLA-Q-Mix was purified by Qiagen DNA Purification kit (see the instructions for specific purification steps), and 200 ul of DNA was purified, and HLA-Q- was determined by Nanodrop 8000 (Thermo Fisher Scientific). The Mix DNA concentration was 48 ng/ul.

4. Interruption of PCR products and construction of Illumina GA PCR-Free sequencing gene library (1) DNA interruption

A total of 5 ug of DNA from the purified HLA-Q-Mix was disrupted on Covaris S2 (Covaris) using Covaris microTube with AFA fiber and Snap-Cap. The breaking conditions are as follows:

Frequency sweeping

(2) Purification after interruption

All the interrupted products of HLA-Q-Mix were recovered and purified by QIAquick PCR Purification Kit, and dissolved in 37.5 ul of EB (QIAGEN Elution Buffer);

(3) End repair reaction

The DNA end-repairing reaction was carried out on the purified product, and the system was as follows (reagents were purchased from Enzymatics):

The reaction conditions were: a temperature bath at 20 ° C for 30 minutes in a Thermomixer (Eppendorf).

The reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 μl of EB (QIAGEN Elution Buffer).

(4) Adding A reaction at the 3' end

In the previous step, the 3' end of the DNA was recovered and the A reaction was carried out. The system was as follows (reagents were purchased from Enzymatics):

The reaction conditions were: a temperature bath at 37 ° C for 30 minutes in a Thermomixer.

The reaction product was recovered and purified by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 13 μl of EB solution (QIAGEN Elution Buffer).

(5) Connect Illumina GA PCR-Free gene bank adapter (adaptor)

The product after addition of A was linked to the Illumina GA PCR-Free gene bank adapter, and the system was as follows (reagents were purchased from Illumina):

The reaction conditions were: overnight at a temperature of 16 ° C in a Thermomixer.

The reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water. The DNA concentration was determined by fluorescent quantitative PCR (QPCR) as follows:

(6) Rubber tapping recycling

30 μL of HLA-Mix was recovered using 2% low melting point agarose gel. The electrophoresis conditions were 100 V for 100 minutes. The DNA standard molecular weight reference is a 50 bp DNA ladder from NEB. The gel was recovered to recover a DNA fragment of 350-550 bp in length (Fig. 2). The recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). The purified volume was 32 ul, and the DNA concentration was 18.83 nM by fluorescent quantitative PCR (QPCR).

5. Illumina GA sequencing

According to the QPCR test results, 10 pmol of DNA was sequenced using the Illumina GA PE-100 program. The specific procedure is described in the Illumina GA operating instructions (Illumina GAIIx).

6. Analysis of results

The sequencing result of Illumina GA is a series of DNA sequences. By searching the sequence of the positive and negative primers and the primer sequence in the sequencing results, the data of the sequencing results of the PCR products of each exon of HLA-DQB1 are established. Library; locate the sequenced results of each exon by BWA (Burrows-Wheeler Aligner) on the reference sequence of the corresponding exon (reference sequence source: http://www.ebi.ac.uk/imgt/hla/ And constructing a consensus sequence for each database; combining base sequencing quality values and the degree of difference between the sequence and the consensus sequence, screening and sequencing error correction of the sequence; and correcting DNA sequences can be assembled into corresponding sequences of HLA-DQB1 2, 3 exons by sequence overlap and pair (Pair-End linkage) relationships. The screenshot of Figure 3 exemplifies the process of constructing the exon 2 consensus sequence of the HLA-DQB1 locus of sample No. 7.

The DNA sequence of the sequenced HLA-DQB1 2 and 3 exons is aligned with the sequence library of the corresponding exon of HLA-DQB1 in the IMGT HLA professional database, and the 100% match of the sequence alignment results is the corresponding sample. HLA-DQB1 genotype.

For all 94 samples, the results obtained were completely consistent with the original known typing results, and the specific results of samples No. 1-32 are shown in Table 3 below.

Example 2 HLA-DQB1 genotyping using Sanger's legal sequence Sample DNA extraction

Similar to the one described in Example 1, 20 of the 94 samples taken by the KingFisher automatic extractor were known to have HLA genotype DNA.

2. PCR amplification

The DNA extracted by the above KingFisher automatic extractor was used as a template, and the PCR primers of Q-F2 and Q-R2, Q-F3 and Q-R3 were single-tube PCR amplification, and the PCR procedures of each pair of primers were as follows:

96 ° C 2 minutes

95°C 30 seconds → 56°C 30 seconds → 72°C 20 seconds (35 cycles)

15 ° C standing

The PCR reaction system of HLA-Q is as follows:

The PCR product was detected by agarose gel electrophoresis and prepared for purification.

3. PCR product purification

PCR product purification was performed using a millipore purification plate. The basic procedure is to mark the wells to be used on the 96-well PCR product purification plate with a marker, and add 50 ul of ultrapure water to the wells to be used. The remaining holes are adhered to the parafilm, allowed to stand at room temperature for 15 minutes or connected to On the suction filtration system, take off at -10 Pa, 5 minutes. Each time the purification plate is removed from the suction filtration system, the liquid remaining in the liquid discharge port at the bottom of the purification plate is blotted on the absorbent paper.

The PCR product to be purified was centrifuged at 4000 rpm for 1 minute; a lid or a silicone pad of the PCR product to be purified was opened, and 100 ul of ultrapure water was added to each PCR reaction system. Then, the purification plate to be added to the PCR product to be purified is connected to the suction filtration system, the vacuum degree is adjusted to a barometer to display -10 Pa, and the microporous regenerated fiber membrane at the bottom of the purification plate is suction-filtered to have no liquid, and the light is observed without completeness. The liquid surface reflects the luster.

Add 50 ul of ultrapure water or TE to the microporous regenerated fiber membrane to the wells to be purified. At room temperature, use a micro-oscillator to shake the plate for 5 minutes, transfer all the liquid in the corresponding well to the new 96-well. The PCR plate corresponds to the well.

4. Perform sequencing reactions and purify the sequencing reaction products

Sequencing reaction using the above purified PCR product as a template

Condition of sequencing reaction

96 ° C 2 minutes

96°C 10 seconds → 55°C 5 seconds → 60°C 2 minutes (25 cycles)

15 ° C standing

The system for sequencing reactions is:

The sequencing reaction product was purified by the following procedure: The sequencing plate was removed and centrifuged at 3000 g for 1 minute. In a 96-well plate, add 2 ul of 0.125 mol/L EDTA-Na2 solution and 33 μL of 85% ethanol per 5 μL reaction system, cover with a silicone pad, shake well for 3 minutes, and centrifuge at 3000 g for 30 minutes at 4 °C. After the end of the centrifugation, the sequencing plate was taken out, the silicone pad was opened, the sequencing reaction plate was inverted on the absorbent paper, and the mixture was centrifuged until the centrifugal force reached 185 g, and immediately stopped. A 96-well plate was filled with 50 ul of 70% ethanol per well, covered with a silicone pad, shaken for 1.5 minutes, and centrifuged at 3000 g for 15 minutes at 4 °C. The sequencing plate was placed in the dark for 30 minutes and air dried until it had no ethanol smell. Add 10 μL per well to a 96-well plate (8 μL per well in a 384-well plate), seal the membrane with HI-DI, shake for 5 seconds, and centrifuge to 1000 rpm.

5. Sequencing and results analysis

The purified sequencing reaction product was subjected to capillary electrophoresis sequencing on ABI 3730XL, and the sequencing peak map was analyzed by uType software (Invitrogen) (Fig. 5) to obtain HLA typing results. All test results are the same as the original test results (Table 4).

It will be apparent to those skilled in the art that various modifications and changes can be made in the present invention without departing from the scope and spirit of the invention. Other embodiments of the present invention will be apparent to those skilled in the art from this disclosure. The description and the examples are to be considered as illustrative only, and the true scope and spirit of the invention are described in the appended claims.

references

[1]. http://www.ebi.ac.uk/imgt/hla/stats.html.

[2]. Tiercy J M. Molecular basis of HLA polymorphism: implications in clinical transplantation. Transpl Immunol, 2002, 9: 173-180.

[3]. C.Antoine, SM Ller, A.Cant, et al . Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience. 1968-99. The Lancet, 2003, 9357: 553-560.

[4]. HA Erlich, G. Opelz, J. Hansen, et al . HLA DNA Typing and Transplantation. Immunity, 2001, 14: 347-356.

[5]. Lillo R, Balas A, Vicario JL, et al ‧Two new HLA class allele, DPB1*02014, by sequence-based typing. Tissue Antigens, 2002, 59: 47-48.

[6]. WU, DL et al . Comparative analysis of serologic typing and HLA-II typing by micro-PCR-SSP. Di Yi Jun Yi Da Xue Xue Bao, 2002, 22: 247-249.

[7]. Al-Hussein KA, Rama NR, Butt AI, et al . HLA class II sequence based typing in normal Saudi individuals. Tissue Antigens, 2002, 60: 259-261.

[8]. Elaine R. Mardis. The impact of next-generation sequencing technology on genetics. Trends in Genetics. 2008, 24: 133-141.

[9]. D. C. Sayer, D. M. Goodridge. Pilot study: assessment of interlaboratory variability of sequencing-based typing DNA sequence data quality. Tissue Antigens, 2007, 69 Suppl: 66-68.

[10]. Horton V, Stratton I, Bottazzo GF et al . Genetic heterogeneity of autoimmune diabetes: age of presentation in adults is influenced by HLA DRB1 and DQB1 genotypes. Diabetologia, 1999, 42: 608-616.

[11]. CEM Voorter, MC Kikl, EM van den Berg-Loonen et al . High-resolution HLA typing for the DQB1 gene by sequence-based typing. Tissue Antigens, 2008, 51: 80-87.

[12].G. Bentley, R. Higuchi, B. Hoglund et al . High-resolution, high-throughput HLA genotyping by next-generation sequencing. Tissue Antigens, 2009, 74: 393-403.

Figure 1: Electrophoresis results of 94 samples of HLA-DQB1 2+3 exon PCR products. From the electropherogram, the PCR product fragment size is a single band between 250 bp and 500 bp, and lane M is DNA. Standard molecular weight reference (DL 2000, Takara), lanes PI-1 to PI-94 were 94 samples of HLA-DQB1 2+3 exon PCR amplification products, negative control (N) without amplification bands;

Figure 2 shows the DNA gel electrophoresis after HLA-Q-Mix was interrupted, with a tapping area of 350-550 bp. Lane M is a DNA standard molecular weight reference (NEB-50bp DNA Ladder), lane 1 shows a gel map of HLA-Q-Mix before tapping, and lane 2 shows a gel map of HLA-Q-Mix after tapping;

Figure 3 shows a partial screenshot of the sample consistency sequence constructor, illustrating the main flow of data analysis. First, the sequence of the DQB1 locus of the sample is aligned to the reference sequence by the BWA software, and the consensus sequence of the DQB1 2 and 3 exons is constructed, and then the DQB1 2 is determined according to the linkage relationship between the SNPs. , 3 exon haplotype sequences. As shown in the figure: in the 7th sample DQB1 gene sequence 2322-2412 contains 6 heterozygous SNPs, the binding relationship of SNP1-SNP5 can be determined by read1 to be TGTCC, and the linkage relationship of another group of SNP1-SNP5 can be determined by read2. In CCAGT, the linkage relationship of SNP3-SNP6 can be determined by read3 as AGTG, and the linkage relationship of another group of SNP3-SNP6 can be determined by read4 as TCCA. The linkage relationship between SNP and read4 can be determined by the linkage relationship of the above SNPs, and read2 is linked with read3. The complete SNP combinations in this region are: TGTCCA and CCAGTG, the sequences of which correspond to the shaded parts of the DQB1*0303 and DQB1*0602 type sequences. The determination of the linkage relationship of other regions is similar;

Figure 4 exemplarily shows two pairs of PCR primers separately amplifying the HLA-DQB1 locus 2 and 3 exons and simultaneously amplifying the electrophoresis patterns of exons 2 and 3, showing three groups from 7 DNA templates. PCR products, all PCR products are less than 500 bp in length, and the electrophoresis bands are single, with no obvious non-specific bands. Negative control (N) without amplification bands, lane M is DNA standard molecular weight reference (DL 2000, Takara);

Fig. 5 exemplarily shows the results of the uType software analysis of the sequencing peaks of the PCR products of the HLA-DQB1 amplification No. 7 template 2 and exon 3, and the DQB1*03:03 DQB1*06 is displayed in the left result output column: The result of 02 is the same as the original known result of the No. 7 template.

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Claims (14)

A polynucleotide having a sequence as shown in any one of sequence identification number: 1 to sequence identification number: 4. A method for amplifying exon 2 and/or exon 3 of HLA-DQB1, characterized by using sequence identification number: 1 and sequence identification number: 2; and/or sequence identification number: 3 and sequence identification number: 4 PCR amplification was performed. A method of sequencing exons 2 and/or exon 3 of HLA-DQB1 in a sample, comprising the steps of: (1) providing a sample and extracting DNA of the sample; (2) identifying the sequence number: 1 and Sequence identification number: 2; and/or sequence identification number: 3 and a primer pair of sequence identification number: 4 for amplifying the DNA to obtain a PCR product; and (3) sequencing the PCR product. The method of claim 3, wherein the PCR product obtained in the step (2) is further subjected to a purification treatment. The method of claim 3, wherein the sample is a blood sample. The method of claim 5, wherein the blood sample is from a mammal or a human. An HLA-DQB1 genotyping method comprising: (1) performing PCR amplification using a sequence identification number: 1 and a sequence identification number: 2; and/or a sequence identification number: 3 and a sequence identification number: 4 Amplifying the exon 2 and/or exon 3 of HLA-DQB1 of the test sample; and (2) sequencing the amplified exon and comparing the sequencing result with the standard sequence in the database, Thereby determining the genotyping results. The method of claim 3, wherein the ordering is by Sanger sequencing or second generation sequencing. The method of claim 8, wherein the second generation sequencing method is Illumina Solexa or Roche 454. A kit for performing HLA-DQB1 genotyping, the kit comprising sequence identification number: 1 and sequence identification number: 2; and/or sequence identification number: 3 and sequence identification number: 4 primer pairs. A kit according to claim 10, which further comprises an agent for DNA amplification, DNA purification and/or DNA sequencing. The use of the polynucleotide of claim 1 for HLA-DQB1 genotyping. The method of claim 7 of the patent application is for the use of HLA-DQB1 genotyping. The kit of claim 10 is for use in HLA-DQB1 genotyping.