WO2012083506A1 - Hla-dqb1基因分型的方法及其相关引物 - Google Patents

Hla-dqb1基因分型的方法及其相关引物 Download PDF

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WO2012083506A1
WO2012083506A1 PCT/CN2010/002150 CN2010002150W WO2012083506A1 WO 2012083506 A1 WO2012083506 A1 WO 2012083506A1 CN 2010002150 W CN2010002150 W CN 2010002150W WO 2012083506 A1 WO2012083506 A1 WO 2012083506A1
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dqb1
hla
sequencing
seq
pcr
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PCT/CN2010/002150
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English (en)
French (fr)
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李剑
张现东
刘莹
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深圳华大基因科技有限公司
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Priority to CN201080070819.5A priority Critical patent/CN103270170B/zh
Priority to PCT/CN2010/002150 priority patent/WO2012083506A1/zh
Priority to MYPI2012005591A priority patent/MY173793A/en
Priority to CA2803940A priority patent/CA2803940C/en
Priority to BR112012032586-8A priority patent/BR112012032586B1/pt
Priority to SG2012096616A priority patent/SG186876A1/en
Priority to US13/807,660 priority patent/US9957564B2/en
Priority to AU2011274090A priority patent/AU2011274090B2/en
Priority to KR1020137002332A priority patent/KR101709826B1/ko
Priority to EP11800190.8A priority patent/EP2599877B1/en
Priority to PCT/CN2011/076688 priority patent/WO2012000445A1/zh
Priority to RU2013103795/10A priority patent/RU2587606C2/ru
Priority to JP2013516983A priority patent/JP5968879B2/ja
Priority to DK11800190.8T priority patent/DK2599877T3/da
Priority to TW100148364A priority patent/TW201300528A/zh
Publication of WO2012083506A1 publication Critical patent/WO2012083506A1/zh
Priority to HK13112600.2A priority patent/HK1185115A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • HLA Human leukocyte antigen
  • HLA-I molecules and HLA-steroids are mainly related to immune rejection
  • HLA-m Molecular function is mainly related to the synthesis of immune-related partial complement systems and inflammation-related factors. Studies have found that the higher the degree of HLA-related gene matching between donor and recipient, the higher the resolution and the longer the graft survival time.
  • HLA-DQB1 belongs to the class of HLA-steroids, and its gene is about 6800 bp in length and consists of 5 introns and 6 exons. In 1996, it was discovered that both the recipient and the recipient
  • HLA-DQB1 genotype matches or not has an effect on the survival rate of the graft.
  • DQB1 locus and dilated cardiomyopathy have been found. And prognosis related.
  • PCR-SSP sequence-specific primer polymerase chain reaction
  • PCR-SSO polymerase chain reaction oligonucleotide probe hybridization
  • PCR-SBT polymerase chain reaction
  • HLA-SSP The principle of HLA-SSP is to design a set of allele-specific primers, 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 the PCR product (gene DNA to be detected) to 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 new alleles.
  • HLA-SBT Sequence-based typing
  • 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 directly determining the nucleic acid sequence by PCR-amplified DNA product, thereby judging the HLA genotype.
  • the high-resolution typing method in addition to the original intuitive, high-resolution and ability to detect new alleles, also features single-molecule sequencing, experimental process cartridges, high throughput and low cost.
  • the DNA length of the sequencing library that can be used for the new sequencing technology can not be too long (the current maximum length of Illumina Solexa is 700 bp), and The new sequencing technology is generally short, and the current Illumia GA bidirectional read length can reach 300bp.
  • the present invention provides PCR primers for amplifying exons 2 and/or exon 3 of HLA-DQB1, which are the SEQ shown in Table 1. ID NO: 1-4.
  • the PCR primers have good conservation and specificity, and can cover the full length sequences of HLA-DQB1 sites 2 and 3, and the PCR products are less than 700 bp in length, which meets the requirements of normal Illumina Solexa sequencing.
  • the primers of the present invention are also suitable for Sanger sequencing.
  • the present invention also provides a novel method for amplifying exon 2 and/or exon 3 of HLA-DQB1, characterized in that PCR amplification is performed using the amplification primer pair of the present invention, and the sequence of the amplification primer pair is shown.
  • Table 1 The sequence of the amplification primer pair is shown.
  • the method of the present invention is particularly advantageously used for HLA-DQB1 genotyping.
  • the method using the method of the present invention and the product of the amplification primer are controlled between 300 and 400 bp, the Illumina Solexa sequencing technology can be utilized for further typing. HLA-SBT.
  • a method of sequencing exons 2 and/or exon 3 of HLA-DQB1 in a sample comprising the steps of:
  • sequencing the PCR product which may be a second generation sequencing method, such as Illumina Solexa or Roche454
  • the invention provides an improved HLA-DQB1 genotyping method, the method comprising:
  • the sequencing method may be Sanger sequencing, or may be a second generation sequencing method, for example, the second generation sequencing method
  • the present invention provides a kit for performing HLA-DQB1 genotyping, the kit comprising the PCR amplification primer pair of the present invention.
  • the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.
  • the amplification primer pair and the genotyping method provided by the present invention can be amplified
  • FIG. 1 is an electrophoresis result 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, wherein lane M is DNA.
  • the 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, and the negative control (N) had no amplification bands.
  • Figure 2 shows the DNA gel electrophoresis after HLA-Q-Mix is interrupted, with a 350-550 bp region.
  • Lane C is the DNA standard molecular weight reference (EB-50bp DNA Ladder)
  • lane 1 shows the HLA-Q-Mix gel before tapping
  • lane 2 shows the HLA-Q-Mix gel after tapping.
  • Figure 3 shows a partial screenshot of the sample consistency sequence constructor, which illustrates the main flow of data analysis.
  • the sequencing sequence of the DQB1 locus of the sample is aligned to the reference sequence by BWA software to construct a consensus sequence of the DQB1 2,3 exon of the sample, and then DQB1 2 is determined according to the linkage relationship between the SNPs. 3 exon haplotype sequence.
  • readl can determine the linkage relationship of SNP1-SNP5 to TGTCC, and the binding relationship of another set of SNP1-SNP5 can be determined by read2 CCAGT, the binding relationship of SNP3-SNP6 can be determined by read3 to 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 maps of exon 2 and exon 3 products, showing three groups from 7 DNA templates.
  • PCR products all PCR products were less than 500 bp in length, and the electrophoresis bands were single, with no obvious non-specific bands.
  • Negative control (N) No amplification bands, lane M is the DNA standard molecular weight reference (DL 2000, Takara).
  • Figure 5 exemplarily shows the results of analysis of the sequencing peaks of the PCR products of HLA-DQB1 amplified No. 7 template 2 and exon 3 by uType software.
  • the left result output column shows DQB1*03:03 DQB1*06:
  • the result of 02 is the same as the original known result of the No. 7 template.
  • the present invention employs the following method to design a PCR primer pair that amplifies the exon 2 and/or exon 3 of HLA-DQB1, downloads all the latest HLA-DQB1 gene sequences from the IMGT/HLA Internet site, and stores them on a local disk as HLA-
  • the DQB1 data set was simultaneously planted with all the latest non-HLA-DQB1 HLA-II gene sequences as comparative data sets.
  • the two data sets were compared, and the conserved and specific sequences of each gene locus were searched for both ends and inside of exons 2 and 3, and the designed PCR primer sequences were compared with human whole genome sequences for homology.
  • PCR primers try to ensure primer 3, which is specific at the end, and ensure that the primers amplify the specificity of HLA-DQB1 gene.
  • the length of the PCR product is less than 700 bp, and the annealing temperature of the positive and negative primers is basically the same.
  • the DNA amplification method, the method of extracting DNA from the sample, and the method of DNA described in the present invention can be selected by those skilled in the art according to the specific circumstances.
  • the method of DNA sequencing can be carried out by a person skilled in the art according to a conventional method or according to the instruction manual of the sequencing instrument.
  • a primer primer with a primer primer sequence of 5 can be used, and the amplified PCR product can be interrupted, and the product is terminated after the disruption. Repair and connect deoxyadenosine (A) at its 3' end, then connect different PCR-free linkers.
  • A deoxyadenosine
  • a sequence of tags is attached to the front end of the amplification primer to achieve simultaneous sequencing of multiple samples.
  • a primer primer can be synthesized by adding a primer index sequence at the 5' end of the PCR primer in combination with PCR-index/barcode technology, and a unique primer label is introduced for each sample during the PCR process.
  • primer labels are designed to vary depending on the experimental platform being used.
  • the present invention mainly considers the following points when designing primer tags: 1: Avoid more than 3 (including 3) single-base repeat sequences in the primer tag sequence, 2: All primer tags The total content of base A and base C in the same site is between 30% and 70% of the total base content. 3: The GC content of the primer tag sequence itself is
  • PCR-Free library adaptor refers to a designed base that functions to assist in the immobilization of DNA molecules on a sequencing chip and to provide a binding site for universal sequencing primers.
  • PCR-Free library linkers can pass DNA. The ligase directly ligates it to both ends of the DNA fragment in the sequencing library, and the introduction process of the linker is called PCR-Free library linker because there is no PCR involved.
  • Adapter or “library adapter” tag technology refers to the addition of different library linkers to multiple sequencing libraries (different library linkers have different composition sequences, and different sequences are called adaptor tags.
  • a tag sequencing library thereby enabling a plurality of different tag sequencing libraries to be mixed and sequenced, and finally a library tagging technique in which the sequencing results of each tag sequencing library can be distinguished from each other.
  • a PCR-FREE library linker is used in the present invention from ILLUMIA.
  • the Sanger method and the second generation sequencing method were 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 factoring with second generation sequencing technology (Illumina Solexa)
  • DNA was extracted from a blood sample (Chinese Hematopoietic Stem Cell Donor Database, also known as "Chinese Bone Marrow Bank") of 94 known HLA-SBT typing results using a KingFisher Automated Extractor (Thermo Corporation, USA).
  • the main steps are as follows: Take out the 6 deep-hole plates and one shallow-hole plate of the Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them. Place all the well-added well plates as required. Corresponding position, select the program "Bioeasy_200 l Blood DNA_KF.msz,, program, press "star” to execute the program for nucleic acid extraction. After the program is finished, collect 100 ⁇ of the eluted product in the plate Elution as the extracted DNA. . 2.
  • Different PCR tag primers can be made by synthesizing PCR primers with different primer tags at 5, such that different PCR tag primers can be used for different samples, the PCR primers are for exons 2 and 3 of HLA-DQB1.
  • the primers were amplified by PCR. Thereafter, a primer tag is introduced at both ends of the PCR product by a PCR reaction, thereby specifically labeling PCR products from different samples.
  • each set of PCR primers consisting of PCR primers for exon 2 or 3 of HLA-DQB1 (Table 1) and a pair of bidirectional primer tags (Table) 2) Composition, wherein each of the forward PCR primers has a forward primer label attached to a pair of primer tags at the 5th end, and a reverse primer label of a pair of primer tags attached to the 5th end of the reverse PCR primer.
  • Primer tags were added directly to the 5' end of the PCR primers during primer synthesis, and the primers were 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.
  • the DQB1 2,3 exons of each sample were amplified in the same reaction well.
  • Two negative controls without template addition were set in the plate, and the primers used for the negative control corresponded to PI-95 and PI-96.
  • the PCR reaction system of HLA-DQBl is as follows:
  • PInf-Q-F2/3 represents primer 5
  • PInf-QR 2/3 represents the primer 5
  • the end has the nth
  • the reverse primer label sequence of the R primer of HLA-DQB1 here n ⁇ 96
  • each sample corresponds to a specific set of PCR primers.
  • FIG. 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).
  • the DNA end-repairing reaction was carried out on the purified product, and the system was as follows (reagents were purchased from Enzymatics):
  • the product purified in the previous step was 37.5 ⁇ lOx polynucleotide kinase buffer ( ⁇ 904) 5 ⁇
  • the reaction conditions were: under a temperature of 20 Torr, in a Thermomixer (Eppendorf) bath for 30 minutes.
  • reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 ⁇ M of Q (QIAGEN Elution Buffer).
  • the DNA was recovered in the previous step, and the end was added with A reaction.
  • the system was as follows (reagents were purchased from Enzymatics):
  • the reaction conditions were: a bath at 37 ° C in a Thermomixer for 30 minutes.
  • reaction product was purified by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 13 ⁇ M of hydrazine solution (QIAGEN Elution Buffer).
  • 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 ⁇ l of deionized water.
  • the DNA concentration was detected by real-time PCR (QPCR). as follows:
  • HLA-Mi 30 ⁇ L of HLA-Mi was recovered with 2% low melting point agarose gel.
  • the electrophoresis conditions were 100V for 100 minutes.
  • the DNA standard molecular weight reference is NEB's 50 bp DNA ladder.
  • the tapping gel recovers DNA fragments ranging from 350-550 bp in length (Fig. 2).
  • the gel recovery product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). The purified volume was 32 ⁇ l, and the DNA concentration was 18.83 nM by real-time PCR (QPCR).
  • the sequencing result of Illumina GA is a series of DNA sequences.
  • a database of sequencing results of each exon PCR product corresponding to each primer label HLA-DQB1 is established; (Burrows-Wheeler Aligner) locate the sequencing results of each exon on the reference sequence of the corresponding exon (reference sequence source: http: ⁇ www.ebi.ac.uk/imgt/hla/), and build each database Consensus sequence; combined with the quality of the base sequencing and the difference between the sequencing sequence and the consensus sequence, screening and sequencing error correction of the sequencing sequence; and the corrected DNA sequence through sequence overlap and linkage
  • the (pair-End linkage) relationship can be assembled into the corresponding sequence of the HLA-DQB1 2,3 exon.
  • the screenshot of Figure 3 exemplifies the process of constructing the exon 2 consensus sequence for the HLA-DQB1 site of sample No. 7.
  • the DNA sequence of the sequenced HLA-DQB1 2,3 exon is aligned with the sequence database of the corresponding exon of HLA-DQB1 in the IMGT HLA professional database.
  • the 100% match of the sequence alignment results is the HLA of the corresponding sample. DQB1 genotype.
  • PCR primers were amplified by Q-F2 and Q-R2, Q-F3 and Q-R3, 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 Cycle); 15 °C ⁇ .
  • the PCR reaction system of HLA-Q is as follows:
  • the PCR product was detected by agarose gel electrophoresis and prepared for purification.
  • the PCR product was purified using a millipore purification plate.
  • the basic steps are: Mark the required wells on the 96-well PCR product purification plate with a marker, add 50 ⁇ M ultrapure water to the wells to be used, attach the remaining pores to the parafilm, let stand for 15 minutes at room temperature or connect to the pump. On the filter system, take -10 Pa, remove for 5 minutes. Each time the purification plate is removed from the suction filtration system, the liquid remaining in the bottom drain of the purification plate is blotted on the absorbent paper.
  • the PCR product to be purified was centrifuged at 4000 rpm for 1 minute; the lid or silica gel pad of the PCR product to be purified was opened, and 100 ⁇ M of ultrapure water was added to each PCR reaction system. Then, the purification plate to which the PCR product to be purified is added 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.
  • the sequencing reaction was carried out by using the above purified PCR product as a template.
  • the sequencing reaction conditions were: 96 "C 2 minutes; 96 ° C 10 seconds 55 ° C 5 seconds 60 ° C 2 minutes (25 cycles); 15 ° C ⁇ .
  • the system of sequencing reactions is:
  • the sequencing reaction product was purified by the following procedure: The sequencing reaction plate was removed and centrifuged, and 3000 g was centrifuged for 1 minute. Each well of the 96-well plate was added with 0.125 mol/L EDTA-Na2 solution 2 ⁇ , 85% ethanol 33 ⁇ l, covered with a silica gel pad, shaken well for 3 minutes, and centrifuged at 3000 g for 30 minutes under 4* ⁇ . After the end of the centrifugation, the sequencing plate was taken out, the silica gel 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.
  • the purified sequencing reaction product was subjected to capillary electrophoresis sequencing on ABI 3730XL, and the sequencing peak image 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).
  • Heterogeneity of autoimmune diabetes age of presentation in adults is influenced by HLA DRB1 and DQB1 genotypes. Diabetologia, 1999, 42:608-616.

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Description

HLA-DQB1基因分型的方法及其相关引物 技术领域 本发明涉及分子生物学领域， 具体而言， 本发明涉及用于 HLA-DQB1基因分型的方法， 以及所述方法中使用的特异性引物。 背景技术 人类白细胞抗原， 即 HLA(human leukocyte antigen, HLA), 是 迄今为止发现的多态性最高的基因系统之一， 它是调控人体特异性 免疫应答和决定疾病易感性个体差异的主要基因系统， 与同种异体 器官移植的排斥反应密切相关。 根据 HLA基因结构和分布的特点， 分为 HLA- I、 HLA- Π和 HLA-m等三类分子， 其中 HLA- I类分子 和 HLA- Π类分子功能主要与免疫排斥相关， HLA-m类分子功能主 要与免疫相关的部分补体系统以及炎症相关因子等的合成相关。 研 究发现， 移植时， 供受双方的 HLA相关基因匹配程度越高， 分辨率 越高， 移植物的存活时间越长。
HLA-DQB1属于 HLA- Π类分子， 其基因长度 6800bp左右， 由 5个内含子和 6个外显子组成。 1996年， 就发现供受双方的
HLA-DQB1基因型别相配与否对移植物的成活率有影响， 近年来， 研究发现 DQB1位点与扩张性心肌症， 病毒性乙型肝炎， 糖尿病， 银屑病等多种疾病的罹患率及预后相关。
目前国际标准的 HLA分型技术包括 PCR-SSP (序列特异引物聚 合酶链式反应）， PCR-SSO (聚合酶链式反应寡核苷酸探针杂交)和 PCR-SBT (聚合酶链式反应产物直接测序分型）。
HLA-SSP的原理是设计出一整套等位基因组特异性引物， 借助 PCR技术获得 HLA型别特异的扩增产物， 通过电泳分析决定 HLA 型别。 HLA-SSO的原理是设计 HLA型别特异的寡核苷酸序列作为 探针， 把 PCR产物标记， 以 PCR产物 （待检测基因 DNA ) 与探针 杂交。通过检测荧光信号判断 HLA型别。 HLA-SSP和 HLA-SSO的 检测信号均是模拟信号， 分辨率只能到达中低水平且都不能检测新 的等位基因。 HLA-SBT ( Sequence-based typing )分型技术是通过对 HLA相 关基因序列测序， 从而判断 HLA基因型别的高分辨分型方法， 具有 直观、 高分辨且能检测新的等位基因的特点。 但现有的 HLA-SBT 是采用 Sanger法测序 （毛细管微电泳） ， 整个实验流程复杂、 通量 低和实验成本高等缺点使其很难应用于大规模 HLA高分辨分型项 目。
基于以 Illumina Solexa和 Roche 454为代表的第二代测序技术 (以下简称新测序技术） 的 HLA-SBT也是一种通过对 PCR扩增后 的 DNA产物直接测定核酸序列， 从而判断 HLA基因型别的高分辨 分型方法， 其除了原有直观、 高分辨且能检测新等位基因的特点外， 还具有单分子测序、 实验流程筒单、 高通量和低成本的特点。 但与 第一代测序技术（以 Sanger法测序原理为基础的测序技术）相比， 能用于新测序技术测序文库制备的 DNA 长度不能太长 （ 当前 Illumina Solexa的最大适用长度为 700bp )， 且新测序技术读长普遍 偏短， 当前 Illumia GA双向读长最大能达到 300bp。
鉴于新测序技术的特点， PCR产物的长度不宜超过 700bp, 原 基于 Sanger法测序方法的 HLA-SBT的 PCR引物不再适用。 因此， 有必要设计一套保守性和特异性良好且 PCR产物长度满足新测序技术 要求的引物。 发明内容 为了将第二代测序技术用于 HLA-DQB1基因分型，本发明提供了 用于扩增 HLA-DQB1的 2和 /或 3号外显子的 PCR引物， 它们是表 1中显示的 SEQ ID NO: 1-4。 所述 PCR引物具有良好的保守性和 特异性， 并且可覆盖 HLA-DQB1位点 2、 3号外显子全长序列， PCR 产物长度均小于 700bp， 满足正常 Illumina Solexa测序要求。 另外， 本发明的引物还适用于 Sanger法测序。
表 1. 用于扩增 HLA-DQB1相应外显子的 PCR引物:
SEQ ID 引物 引物序列 HLA-DQB1 扩增产 NO: 编号 外显子 长度物
1 Q-F2 GATTCCYCGCAGAGGATTTCG
2号 311bp
2 Q-R2 AGGGGCRACSACGCTCACCTC
3 Q-F3 CCTGTCTGTTACTGCCCTCAGT
3号 339bp
4 Q-R3 GGCCCATAGTAACAGAAACTCAATA 本发明还提供了一种新的 HLA-DQB1的 2和 /或 3号外显子扩增 方法， 其特征在于使用本发明的扩增引物对进行 PCR扩增， 所述扩增 引物对的序列示于表 1。
由于能够通过 PCR反应扩增出 HLA-DQB1的 2和 /或 3号外显子， 因此， 本发明的方法特别有利地可用于进行 HLA-DQB1基因分型。 与 现有的 0LA-DQB1基因分型方法相比， 由于使用本发明的方法和扩增 引物的产物被控制在 300-400bp之间， 因此在进行进一步分型时， 可以 利用基于 Illumina Solexa测序技术的 HLA-SBT。
在本发明的第三方面中， 提供了一种对样品中的 HLA-DQB1的 2和 /或 3号外显子进行测序的方法， 其包括步骤：
1 ) 提供一个样品并提取该样品的 DNA;
2 ) 将表 1中的 PCR引物用于扩增所述 DNA从而得到 PCR产 物， 优选对 PCR产物进行纯化；
3 ) 对所述 PCR产物进行测序，测序方法可以是第二代测序法， 例如 Illumina Solexa或 Roche454„
在本发明的第四方面中， 本发明提供了一种改进的 HLA-DQB1 基因分型方法， 所述方法包括：
1) 使用本发明的 PCR扩增引物对扩增待测的 HLA-DQB1的 2 和 /或 3号外显子，
2)对扩增出的外显子进行测序， 并将测序结果与数据库中的标准 序列进行比较， 从而确定基因分型结果。 其中测序方法可以是 Sanger 测序法， 或者可以是第二代测序法， 所述第二代测序方法例如
Illumina Solexa或 Roche 454.
另一方面， 本发明还提供了一种用于进行 HLA-DQB1基因分型的 试剂盒， 所述试剂盒中包含本发明的 PCR扩增引物对。 在一个实施方 案中， 所述试剂盒还包含其他试剂， 例如用于 DNA扩增、 DNA纯化和 /或 DNA测序的试剂。
应用本发明提供的扩增引物对和基因分型方法， 能够在扩增
HLA-DQB1的 2和 /或 3号外显子的基础上进行基因分型。 相对于现 有技术而言， 该分型方法利用了 Illumina Solexa测序技术， 该技术具 有可高通量、 低成本地获得高分辨的 HLA分型结果的特点。 附图说明 图 1为 94个样本 HLA-DQB1 2+3外显子 PCR产物电泳结果， 从 电泳图上看， PCR产物片段大小为位于 250bp-500bp之间的单一条带， 其中泳道 M是 DNA标准分子量参照物 （DL 2000, Takara公司） ， 泳 道 PI-1至 PI-94为 94个样本的 HLA-DQB1 2+3外显子 PCR扩增产物， 阴性对照 （N )无扩增条带。
图 2显示对 HLA-Q-Mix打断后 DNA电泳凝胶割胶的情况， 割 胶区域为 350-550bp区域。 其中泳道 M是 DNA标准分子量参照物 ( EB-50bp DNA Ladder ) , 泳道 1示出割胶前 HLA-Q-Mix的胶 图， 泳道 2示出割胶后 HLA-Q-Mix的胶图。
图 3示出 7号样本一致性（consensus )序列构建程序部分截图， 示例性说明了数据分析的主要流程。 首先， 该样本的 DQB1位点的 测序序列通过 BWA软件比对到参考序列上，构建出该样本 DQB1 2,3 外显子的一致性序列，再根据 SNP之间的连锁关系来确定 DQB1 2,3 外显子单体型序列。如图所示：在 7号样本 DQB1基因序列 2322-2412 区域含有 6个杂合 SNP, 由 readl可确定 SNP1-SNP5的连锁关系为 T-G-T-C-C，由 read2 可确定另一组 SNP1-SNP5 的连锁关系为 C-C-A-G-T, 由 read3可确定 SNP3-SNP6的连锁关系为 A-G-T-G，由 read4 可确定另一组 SNP3-SNP6 的连锁关系为 T-C-C-A,通过上述 SNP的连锁关系可确定 readl与 read4连锁， read2与 read3连锁， 在此区域完整的 SNP组合为： T-G-T-C-C-A和 C-C-A-G-T-G, 其序 列对应 DQB1*0303和 DQB1*0602型别序列的阴影部分。 其它区域 的连锁关系的判定与此类似。
图 4示例性显示了两对 PCR引物分别单独扩增 HLA-DQB1位 点 2和 3号外显子和同时扩增 2和 3号外显子产物电泳图， 图中显 示来自 7个 DNA模板的三組 PCR产物，所有 PCR产物长度均小于 500bp, 且电泳条带单一， 无明显的非特异性条带。 阴性对照 （N ) 无扩增条带，泳道 M是 DNA标准分子量参照物 （DL 2000， Takara 公司 ) 。
图 5示例性显示了 HLA-DQB1扩增 7 号模板 2和 3号外显子的 PCR 产物的测序峰图经 uType 软件分析的结果， 左侧结果输出栏中显示 DQB1*03:03 DQB1*06:02的结果， 与 7号模板原已知结果相同。 具体实施方式 下面将结合实施例对本发明的实施方案进行详细描述。 本领域技 术人员应理解， 下列实施例仅用于说明本发明， 而不应视为限定本发 明的范围。
本发明采用如下方法设计扩增 HLA-DQB1的 2和 /或 3号外显子 的 PCR引物对，从 IMGT/HLA因特网站点下载所有最新 HLA-DQB1 基因序列， 然后保存到本地磁盘中做为 HLA-DQB1数据集； 同时下栽 所有最新非 HLA-DQB1的 HLA-II类基因序列做为比较数据集。 将两 数据集进行比较， 在 2， 3号外显子两端和内部寻找各基因位点保守和 特异序列，并将设计的 PCR引物序列与人类全基因组序列进行同源性 比较。 在设计 PCR引物时尽量保证引物 3，末端特异， 确保引物扩增 HLA-DQB1基因的特异性。 同时使 PCR产物的长度小于 700bp, 且正 反引物的退火温度基本保持一致。
将满足设计要求的多对候选 HLA-DQB1引物用于扩增少数具有 HLA-DQB1常见血清型的模板 DNA,从中筛选出保守性和特异性最好 的， 分别用于扩增 2和 3号外显子的 2对 HLA-DQB1的 PCR引物。
本发明中涉及的 DNA扩增方法、 从样品提取 DNA的方法、 DNA 述方法可以由本领域技术人员'根据^具体情况进行选择。 DNA测序 的方法， 本领域技术人员可以根据常规的方法进行， 或者根据测序仪 器的使用说明书进行。
例如， 在利用二代测序技术进行测序过程中， 可以使用 5，末端添加 引物标签（ primer index )序列的标签引物进行， 可以将扩增后的 PCR 产物进行打断， 并且打断后产物进行末端修复并在其 3，端连接脱氧腺苷 ( A ) ， 然后连接不同的 PCR-free接头。
在扩增引物前端连接一段标签序列是为了实现同时对多个样品进行 测序。 具体而言， 可以结合 PCR-index/barcode技术， 通过在 PCR引物 的 5，末端添加引物标签（ primer index )序列合成标签引物， 在 PCR过 程中对每个样本引入独特的引物标签。这样，在利用第二代 DNA测序技 术检测过程中， 除 PCR环节必须逐个样本处理外， 其它实验环节可把多 个样本混在一起同时处理， 最终每个样本的检测结果可以通过其独特的 引物标签序列找回。引物标签的设计根据所应用的实验平台不同而不同， 考虑 Illumina GA测序平台本身的特点， 本发明在设计引物标签时主要 考虑了以下几点： 1: 引物标签序列中避免 3个以上（包括 3个）单碱基 重复序列， 2: 所有引物标签的同一位点中碱基 A和碱基 C的总含量占 所有碱基含量的 30%-70%之间， 3: 引物标签序列本身的 GC含量在
40-60%之间， 4: 引物标签之间序列差异度大于 4个碱基， 5: 引物标签 序列中避免出现与 Illumina GA测序引物相似度高的序列， 6: 减少引物 标签序列添加到 PCR引物上后，对 PCR引物造成的严重发卡（ hairpin )， 二聚体（dimer ) 情况的出现。
术语" PCR-Free文库接头（adapter ) "是指经设计的一段碱基， 其主 要作用是辅助固定 DNA分子在测序芯片上以及提供通用测序引物的结 合位点， PCR-Free文库接头可以通过 DNA连接酶将其直接连接至测序 文库中的 DNA片段两端，接头的导入过程因为没有 PCR的参与， 因此称 作 PCR-Free文库接头。 "接头（ adapter ) "或"文库接头（ library adapter )" 标签技术是指通过对多个测序文库添加不同文库接头 （不同文库接头的 组成序列不同， 序列不同的部分称为接头标签（adapter index ) ) ， 构 建标签测序文库， 从而可实现多个不同标签测序文库混合测序， 且最终 各个标签测序文库的测序结果可相互区分的一种文库标签技术。 例如， 本发明实施例中使用 PCR-FREE文库接头来自 ILLUMIA。
在如下的实施例中，用筛选出的 2对 PCR引物，对 94例已知 HLA 常见基因型别的血样进行 HLA-DQB1位点 PCR扩增， 扩增产物经
Sanger法和第二代测序方法进行测序。 将测序结果用于 HLA-DQB1 分型， 并通过与原分型结果比较来验证 PCR引物的保守性和特异性。
实施例 1: 用第二代测序技术（ Illumina Solexa )进行 HLA-DQB1 因分型
1. 样本提取
使用 KingFisher自动提取仪（美国 Thermo公司） 从 94份已知 HLA-SBT分型结果的血样（中国造血干细胞捐献者资料库，也称为 "中 华骨髓库"） 中提取 DNA。 主要步骤如下： 取出 6个 Kingfisher自动 提取仪配套的深孔板及 1个浅孔板， 根据说明书分别加入一定量配套 的试剂并做好标记，将所有已加好试剂的孔板按要求置于相应的位置， 选定程序" Bioeasy_200 l Blood DNA_KF.msz，，程序，按下" star"执行该 程序进行核酸提取。 程序结束后收集 plate Elution中的 100 μΐ左右的 洗脱产物即为提取的 DNA。 2. PCR扩增
通过合成在 5，末端具有不同引物标签的 PCR引物制作不同的 PCR标签引物， 这样不同的 PCR标签引物可以用于不同的样本， 所 述 PCR引物是针对 HLA-DQB1的 2和 3号外显子的 PCR扩增引物。 其后通过 PCR反应在 PCR产物两端引入引物标签， 从而特异地标记 了来自不同样本的 PCR产物。
以 94套 PCR标签引物来分别扩增 94份 DNA样本，每套 PCR标 签引物由用于扩增 HLA-DQB1的 2或 3号外显子的 PCR引物（表 1 ) 和一对双向引物标签（表 2 )组成， 其中每个正向 PCR引物的 5，末端 上连接一对引物标签的正向引物标签，而反向 PCR引物的 5，末端上连 接一对引物标签的反向引物标签。 引物标签在引物合成时直接添加在 PCR引物的 5，末端， 引物由上海英潍捷基（ Invitrogen )公司合成。
把样本提取步骤中所得的 94份 DNA， 依次编号 1-94， PCR反应 在 96孔板中进行， 每个样本的 DQB1 2，3外显子在同一个反应孔中进 行扩增。 板内设置两个不添加模板的阴性对照， 阴性对照所用引物对 应标签号为 PI-95和 PI-96。 实验的同时， 记录下每对引物标签对应的 样本编号信息。
表 2. 引物标签的相关信息:
引物标 对应 96
正向引物标签 反向引物标签 对应模板 签编号 孔板位置
PI-1 TCGCAGACATCA TGACACGATGCT A1 1
PI-2 TACATCGCACTA TACAGATGCTGA A2 2
PI-3 CTCGATGAGTAC ACGTCTAGACAC A3 3
PI-4 TCTGTATACTCA TGCTGTAGTGAC A4 4
PI-5 TATCTGCTCATA AGATATCGAGCT A5 5
PI-6 TACATGCTGAGC ACGTGTCTATCA A6 6
PI-7 TCATATCGCGAT AGATCGTATAGC A7 7
PI-8 ACAGATGCACGC ATCTCGTGACAG A8 8
PI-9 TAGATCGTACAT ACTAGTACACGC A9 9
PI-10 ACTACACGTCTC ATAGTCACGCGT A10 10
PI-11 AGACTCGCGTAT TACTAGCTGACG All 11
PI-12 ATACTAGTGCTC TGTATCGTGCTC A12 12
PI-13 CACGATGACATC TAGTGAGCGCAC B1 13
PI-14 TGCTGTCTCGAG CATAGCAGTGTC B2 14
PI-15 TGTGCTCGAGTC TCTGATCGAGCA B3 15
PI-16 CACTCGTACATC AGCGATGCTCAT B4 16
PI-17 CGACGTGCTCGC CGCGTACTGCAG B5 17
PI-18 ACGCATCTATAC CTAGTATCGCAG B6 18
PI-19 CGAGATGACTCT TGTATACACGAT B7 19
PI-20 ACTGTCTCGAGC ACGTAGCGCACA B8 20 69 6Λ VX3XDV3VXOV3 69-Id
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£Ζ iia 3V0XVX30I3XV VXDV3VXV0V01 £Z-ld ζζ oie XV0X3V30XVI3 V0V13X3V303V IZ-\A ιζ 6a VOIV3XDOVX3X 工 VI OJ^IVO MIASTZ00/010ZN3/X3d 90SC80/ZT0Z OAV PI-70 CATACTATCACG TCGTGTCACTAC F10 70
PI-71 CACTATACAGAT CGACACGTACTA Fll 71
PI-72 ATATCGTAGCAT TCGTGATCACTA F12 72
PI-73 TAGTCTATACAT AGACGCTGTCGA Gl 73
PI-74 TGTCACAGTGAC TCATATGATCGA G2 74
PI-75 ATCGACTATGCT CGATCATATGAG G3 75
PI-76 ATACTAGCATCA TCATGCTGACGA G4 76
PI-77 CACTGACGCTCA CACTACATCGCT G5 77
PI-78 TCGCTCATCTAT TAGTACAGAGCT G6 78
PI-79 TGTATCACGAGC ATGATCGTATAC G7 79
PI-80 TACTGCTATCTC CGCTGCATAGCG G8 80
PI-81 CGCGAGCTCGTC ACTCGATGAGCT G9 81
PI-82 TAGAGTCTGTAT TGTCTATCACAT G10 82
PI-83 TACTATCGCTCT TATGTGACATAC Gil 83
PI-84 TAGATGACGCTC TACTCGTAGCGC G12 84
PI-85 TCGCGTGACATC ATCTACTGACGT HI 85
PI-86 ACACGCTCTACT ACAGTAGCGCAC H2 86
PI-87 TACATAGTCTCG CTAGTATCATGA H3 87
PI-88 TGAGTAGCACGC TCGATCATGCAG H4 88
PI-89 TAGATGCTATAC TACATGCACTCA H5 89
PI-90 ATCGATGTCACG CAGCTCGACTAC H6 90
PI-91 ATCATATGTAGC CTCTACAGTCAC H7 91
PI-92 TAGCATCGATAT AGATAGCACATC H8 92
PI-93 TGATCGACGCTC CTAGATATCGTC H9 93
PI-94 TGCAGCTCATAG TACAGACTGCAC H10 94
PI-95 CGACGTAGAGTC CAGTAGCACTAC Hll 阴性对照
PI-96 CACTGTATAGCT CGACTAGTACTA H12 阴性对照
HLA-DQBl的 PCR程序如下：
96 °C 2分钟； 95°C 30秒 30秒" >72°C 20秒 (32个循环）; °C∞。
HLA-DQBl的 PCR反应体系如下：
Figure imgf000010_0001
其中 PInf-Q-F2/3表示引物 5，末端带有第 n号正向引物标签序列 (表 1 ) 的 HLA-DQB1的 F引物， PInf-Q-R 2/3表示引物 5，末端带 有第 n号反向引物标签序列的 HLA-DQB1的 R引物 （此处 n≤96 ) ， 其它依次类推。 且每个样本对应特定的一套 PCR引物。
PCR反应在 Bio-Rad公司的 PTC-200 PCR仪上运行。 PCR完成 后， 取 2 l PCR产物经 1.5%的琼脂糖凝胶电泳检测。 图 1显示了 94 个样本 HLA-DQB1 2+3外显子 PCR产物电泳结果， DNA标准分子量 参照物 （M ) 为 DL 2000 ( Takara公司） 。
3. PCR产物混合和纯化
从 96孔板 HLA-P-DQB1剩余的 PCR产物中 （阴性对照除外）各 取 20 μΐ混合在一个 3 ml的 ΕΡ管中，标记为 HLA-Q-Mix并混合均匀。 从 HLA-Q-Mi 中取 500 μΐ DNA混合物经 Qiagen DNA Purification kit 过柱纯化 （具体纯化步骤详见说明书） ， 纯化所得的 200 μ1 ϋΝΑ， 经 Nanodrop 8000(Thermo Fisher Scientific公司）测定 HLA-Q-Mix DNA 浓度为 48 iig/ l。
4. PCR产物的打断以及 Illumina GA PCR-Free测序文库的构建 1 ) DNA打断
从纯化后的 HLA-Q-Mix中取总量 5 的 DNA 用 Covaris microTube with AFA fiber and Snap - Cap在 Covaris S2(Covaris公司 ) 上打断。 打断条件如下：
频率扫描 ( frequency sweeping )
Figure imgf000011_0001
2 )打断后纯化
将 HLA-Q-Mix的所有打断产物用 QIAquick PCR Purification Kit 回收純化， 分别溶于 37.5 μΐ的 EB ( QIAGEN Elution Buffer ) 中；
3 )末端修复反应
对纯化的产物进行 DNA末端修复反应， 体系如下 （试剂均购自 Enzymatics公司 ) ：
上一步骤纯化的产物 37.5 μΐ lOx多核苷酸激酶緩冲液（Β904 ) 5 μΐ
dNTP混合物 （每种 10 mM ) 2 μΐ
T4 DNA聚合酶 2.5 μΐ
Klenow片段 0.5 μΐ
T4多聚核苷酸激酶 2.5 μΐ 总体积 50 μΐ
反应条件为： 在 20 Ό下， 在 Thermomixer(Eppendorf公司）中温 浴 30分钟。
反应产物经 QIAquick PCR Purification Kit回收纯化， 溶于 34 μΐ 的 ΕΒ ( QIAGEN Elution Buffer ) 中。
4 ) 3，末端加 A反应
上一步回收 DNA的 3，末端加 A反应， 体系如下 （试剂均购自 Enzymatics公司 ) ：
Figure imgf000012_0001
反应条件为： 在 37°C下， 在 Thermomixer中温浴 30分钟。
反应产物经 MiniElute PCR Purification Kit ( QIAGEN公司） 回 收纯化， 溶于 13 μΐ的 ΕΒ溶液（ QIAGEN Elution Buffer ) 中。
5 )连接 Illumina G A PCR-Free文库接头 （adaptor )
加 A后的产物连接 Illumina GA PCR-Free文库接头，体系如下（试 剂均购自 Illumina公司） ：
Figure imgf000012_0002
反应条件为： 在 16°C下， 在 Thermomixer中温浴过夜。
反应产物经 Ampure Beads(Beckman Coulter Genomics)纯化后溶 于 50 μΐ去离子水， 经荧光定量 PCR ( QPCR )检测到 DNA浓度结果 如下:
Figure imgf000013_0001
6 )割胶回收
取 30 μΐ HLA-Mi 用 2%低熔点琼脂糖胶进行回收。 电泳条件为 100V, 100分钟。 DNA标准分子量参照物为 NEB公司的 50 bp DNA ladder. 割胶回收 350-550 bp长度范围的 DNA片段（图 2 ) 。 胶回收 产物经 QIAquick PCR Purification Kit ( QIAGEN公司） 回收纯化， 纯化后体积为 32 μ1， 经荧光定量 PCR ( QPCR )检测到 DNA浓度结 果为 18.83 nM。
5. Illumina GA测序
根据 QPCR检测结果，取 10 pmol DNA用 Illumina GA PE-100程 序测序， 具体操作流程详见 Illumina GA操作说明书 （Illumina GA H x ) 。
6. 结果分析
Illumina GA产出的测序结果是一系列 DNA序列， 通过查找测序 结果中的正反引物标签序列和引物序列， 建立各个引物标签对应样本 HLA-DQB1各外显子 PCR产物测序结果的数据库； 通过 BWA (Burrows-Wheeler Aligner)把各外显子的测序结果定位在相应外显子 的参考序列上 （参考序列来源： http：〃 www.ebi.ac.uk/imgt/hla/ ) , 并 构建各个数据库的一致性（consensus )序列； 结合碱基测序质量值和 测序序列与 consensus序列的差异度，对测序序列进行筛选和测序错误 校正； 以及校正后的 DNA序列通过序列重叠 （overlap )和连锁
( Pair-End连锁） 关系可组装成 HLA-DQB1 2,3外显子相应的序列。 图 3的截图示例性说明了对 7号样品的 HLA-DQB1位点的 2号外显子 一致性序列进行构建的过程。
将所测序的 HLA-DQB1 2,3外显子的 DNA序列与 IMGT HLA专 业数据库中 HLA-DQB1相应外显子的序列数据库比对，序列比对结果 100%匹配的即为对应样本的 HLA-DQB1基因型别。
所有 94个样本， 得到的分型结果与原已知分型结果完全相符， 其 中 1-32号样本的具体结果下表 3中。
表 3. 1-32号样本的分型结果： 样本编号 原 DQB1基因型别 本次 DQB1检测结果 是否相同
1 DQB1*02:02 DQB1*03:01 DQB1*02:02 DQB1*03:01 是
2 DQB1*02:02 DQB1*04:01 DQB1*02:02 DQB1*04:01 是
3 DQB1*05:02 DQB1*02:02 DQB1*05:02 DQB1*02:02 是
4 DQB1*02:02 DQB1*06:03 DQB1*02:02 DQB1*06:03 是
5 DQB1*03:03 DQB1*04:02 DQB1*03:03 DQB1*04:02 是
6 DQB1*05:02 DQB1*03:17 DQB1*05:02 DQB1*03:17 是
7 DQB1*03:03 DQB1*06:02 DQB1*03:03 DQB1*06:02 是
8 DQB1*05:03 DQB1*04:02 DQB1*05:03 DQB1*04:02 是
9 DQB1*04:02 DQB1*06:01 DQB1*04:02 DQB1*06:01 是
10 DQB1*05:01 DQB1*06:10 DQB1*05:01 DQB1*06:10 是
11 DQB1*03:01 DQB1*03:03 DQB1*03:01 DQB1*03:03 是
12 DQB1*05:01 DQB1*05:01 DQB1*05:01 DQB1*05:01 是
13 DQB1*02:02 DQB1*04:02 DQB1*02:02 DQB1*04:02 是
14 DQB1*05:02 DQB1*02:01 DQB1*05:02 DQB1*02:01 是
15 DQB1*02:01 DQB1*06:02 DQB1*02:01 DQB1*06:02 是
16 DQB1*03:03 DQB1*04:01 DQB1*03:03 DQB1*04:01 是
17 DQB1*05:01 DQB1*03:02 DQB1*05:01 DQB1*03:02 是
18 DQB1*03:03 DQB1*06:01 DQB1*03:03 DQB1*06:01 是
19 DQB1*03:03 DQB1*06:10 DQB1*03:03 DQB1*06:10 是
20 DQB1*05:03 DQB1*04:01 DQB1*05:03 DQB1*04:01 是
21 DQB1*05:02 DQB1*04:01 DQB1*05:02 DQB1*04:01 是
22 DQB1*03:01 DQB1*03:03 DQB1*03:01 DQB1*03:03 是
23 DQB1*05:02 DQB1*05:03 DQB1*05:02 DQB1*05:03 是
24 DQB1*05:02 DQB1*03:02 DQB1*05:02 DQB1*03:02 是
25 DQB1*03:03 DQB1*06:01 DQB1*03:03 DQB1*06:01 是
26 DQB1*05:02 DQB1*06:09 DQB1*05:02 DQB1*06:09 是
27 DQB1*02:02 DQB1*06:02 DQB1*02:02 DQB1*06:02 是
28 DQB1*05:02 DQB1*03:01 DQB1*05:02 DQB1*03:01 是
29 DQB1*02:01 DQB1*03:01 DQB1*02:01 DQB1*03:01 是
30 DQB1*06:03 DQB1*06:09 DQB1*06:03 DQB1*06:09 是
31 DQB1*05:02 DQB1*02:02 DQB1*05:02 DQB1*02:02 是
32 DQB1*05:01 DQB1*06:01 DQB1*05:01 DQB1*06:01 是 实施例 2. 用 Sanger法测序进行 HLA-DQB1基因分型
1. 样品 DNA提取
如实施例 1中所述相似， 以 KingFisher自动提取仪提取的 94例 样本中的 20例已知 HLA基因型别的 DNA。
2. PCR扩增
以上述 KingFisher自动提取仪提取的 DNA为模板， 以 Q-F2和 Q-R2、 Q-F3和 Q-R3共 2对 PCR引物分别单管 PCR扩增， 各对引物 PCR程序如下： 96°C 2分钟； 95。C 30秒 56°C 30秒 72。C 20秒 (35 循环）； 15 °C∞。
HLA-Q的 PCR反应体系如下:
Figure imgf000015_0001
PCR产物经琼脂糖凝胶电泳检测后， 准备纯化。
3. PCR产物纯化
利用 millipore纯化板进行 PCR产物纯化。 基本步骤是： 用记号 笔在 96孔 PCR产物纯化板上标记需要使用的孔， 并向需要使用的孔 中加入 50 μΐ超纯水， 剩余孔粘贴封口膜， 室温静置 15分钟或连接到 抽滤系统上， -10帕， 5分钟取下， 每次从抽滤系统上取下纯化板时都 要在吸水纸上吸干残留在纯化板底部排液口的液体。
待纯化 PCR产物离心， 4000 rpm, 1分钟； 打开待纯化 PCR产物 的盖子或硅胶垫，每个 PCR反应体系中加入 100 μΐ超纯水。 然后把加 入待纯化 PCR产物的纯化板连接到抽滤系统上，调节真空度至气压表 显示 -10 Pa，抽滤至純化板底部的微孔再生纤维膜上无液体，光照下观 察， 无完整液面反射光泽。
向有待纯化 PCR产物的孔中加 50 μΐ超纯水或 ΤΕ到微孔再生纤 维膜上； 室温下使用微量振荡器中档振荡纯化板 5分钟， 转移相应孔 内全部液体至新的 96孔 PCR板对应的孔中。
4. 进行测序反应并纯化测序反应产物
以上述纯化后的 PCR产物为模板做测序反应， 测序反应条件是： 96 "C 2分钟； 96°C 10秒 55 °C 5秒 60 °C 2分钟 （25个 循环）； 15°C ∞。
测序反应的体系是：
Figure imgf000015_0002
5*Buffer 0.85 μΐ
水 1.85 μΐ
总体积 5 μΐ
通过以下步骤纯化测序反应产物：取下测序反应板配平，离心 3000 g, 1分钟。 96孔板每 5 μΐ反应体系加 0.125 mol/L EDTA-Na2溶液 2 μΐ, 85%乙醇 33 μ1， 盖上硅胶垫， 充分振荡 3分钟， 在 4*Ό下以 3000 g离 心 30分钟。 离心结束后取出测序板， 打开硅胶垫， 将测序反应板倒置 吸水纸上，倒离心至离心力达到 185 g时立即停止。 96孔板每孔加 70% 乙醇 50 μ1， 盖上硅胶垫， 振荡 1.5分钟， 在 4。C下以 3000 g离心 15分 钟。 测序反应板置避光通风处 30分钟， 风干至无乙醇气味。 96孔板 每孔加 ΙΟ μΙ ( 384孔板每孔加 8 μ1 ) HI-DI甲酰胺， 盖封口膜， 振荡 5 秒后离心至 1000 rpm。
5. 测序和结果分析
纯化后的测序反应产物在 ABI 3730XL上进行毛细管电泳测序， 测序峰图经过 uType软件 (Invitrogen)分析（图 5)，得到 HLA分型结果。 全部检测结果与原有检测结果相同 （表 4 ) 。
表 4. 本次分型结果与样本原来分型结果对比:
Figure imgf000016_0001
对于本领域技术人员显而易见的是， 在不背离本发明的范围和精 神的前提下可对本发明进行各种修改和变动。 通过考虑本发明在此所 公开的说明书和实例， 本发明的其他实施方案对本领域技术人员来说 是显而易见的。 本说明书和实施例应仅看作示例性用途， 本发明真正 的范围和精神在所附的权利要求书中说明。
参考文献
[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, S.Muller, A.Cant, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodenciencies:
report of the European experience. 1968-99. The Lancet,
2003,9357:553-560.
[4]. H. A. 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， D. L. 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 K A, Rama N R, Butt A I， 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 G. F. 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]. C.E.M. Voorter, M.C. Kikl, E.M. van den Berg-Loonen et al. High-resolution HLA typing for the DQBl 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.

Claims

权 利 要 求 书
1. 具有 SEQ ID NO: 1至 SEQ ID NO: 4任一项所示序列的多核 苷酸。
2. 一种 HLA-DQB1的 2和 /或 3号外显子扩增方法， 其特征在 于使用 SEQ ID NO: 1和 SEQ ID NO: 2; 以及 /或者 SEQ ID NO: 3和 SEQ ID NO: 4的引物对进行 PCR扩增。
3. 一种对样品中 HLA-DQB1的 2和 /或 3号外显子进行测序的 方法， 其包括步骤：
1 ) 提供一个样品并提取该样品的 DNA;
2 ) 将 SEQ ID NO: 1和 SEQ ID NO: 2; 以及 /或者 SEQ ID NO: 3和 SEQ ID NO: 4的引物对用于扩增所述 DNA从而得到 PCR产物， 优选对 PCR产物进行纯化；
3 ) 对所述 PCR产物进行测序。
4. 权利要求 3的方法， 其中所述样品是血样。
5. 权利要求 4的方法， 其中所述血样来自哺乳动物或人。
6. 一种 HLA-DQB1基因分型方法， 所述方法包括：
1)使用 SEQ ID NO: 1和 SEQ ID NO: 2; 以及 /或者 SEQ ID NO: 3和 SEQ ID NO: 4的引物对进行 PCR扩增， 扩增待测样本的
HLA-DQB1的 2和 /或 3号外显子；
2)对扩增出的外显子进行测序， 并将测序结果与数据库中的标准 序列进行比较， 从而确定基因分型结果。
7. 权利要求 3或 6的方法， 其中所述测序是通过 Sanger测序法 或第二代测序法。
8. 权利要求 7的方法， 其中所述第二代测序方法是 Illumina Solexa或 Roche454。
9. 一种用于进行 HLA-DQB1基因分型的试剂盒， 所述试剂盒中 包括 SEQ ID NO: 1和 SEQ ID NO: 2; 以及 /或者 SEQ ID NO: 3和 SEQ ID NO: 4的引物对，所述试剂盒优选还包含用于 DNA扩增、 DNA 纯化和 /或 DNA测序的试剂。
10. 权利要求 1的多核苷酸、 权利要求 6的方法或权利要求 9的试 剂盒用于 HLA- DQB1基因分型的用途。
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