WO2012083505A1 - Method for hla-c genotyping and related primers thereof - Google Patents

Method for hla-c genotyping and related primers thereof Download PDF

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WO2012083505A1
WO2012083505A1 PCT/CN2010/002149 CN2010002149W WO2012083505A1 WO 2012083505 A1 WO2012083505 A1 WO 2012083505A1 CN 2010002149 W CN2010002149 W CN 2010002149W WO 2012083505 A1 WO2012083505 A1 WO 2012083505A1
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seq
hla
sequencing
pcr
dna
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PCT/CN2010/002149
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French (fr)
Chinese (zh)
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李剑
陈仕平
张彩芬
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深圳华大基因科技有限公司
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Priority to CN201080070820.8A priority Critical patent/CN103261438B/en
Priority to PCT/CN2010/002149 priority patent/WO2012083505A1/en
Priority to PCT/CN2011/076688 priority patent/WO2012000445A1/en
Priority to AU2011274090A priority patent/AU2011274090B2/en
Priority to US13/807,660 priority patent/US9957564B2/en
Priority to CA2803940A priority patent/CA2803940C/en
Priority to KR1020137002332A priority patent/KR101709826B1/en
Priority to DK11800190.8T priority patent/DK2599877T3/en
Priority to SG2012096616A priority patent/SG186876A1/en
Priority to JP2013516983A priority patent/JP5968879B2/en
Priority to EP11800190.8A priority patent/EP2599877B1/en
Priority to BR112012032586-8A priority patent/BR112012032586B1/en
Priority to MYPI2012005591A priority patent/MY173793A/en
Priority to RU2013103795/10A priority patent/RU2587606C2/en
Priority to TW100148367A priority patent/TWI542696B/en
Publication of WO2012083505A1 publication Critical patent/WO2012083505A1/en
Priority to HK13112599.5A priority patent/HK1185114A1/en

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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

Definitions

  • HLA human leucocyte antigen
  • Rejection-related, HLA-m class molecular function is mainly related to the synthesis of immune-related partial complement system and inflammation-related factors; clinical transplantation studies found that graft long-term survival rate and HLA-I and HLA of both donors and recipients - The degree of matching of the II molecules is positively correlated. The higher the degree of matching between the donor and recipient HLA molecules, the higher the long-term survival rate of the graft.
  • the HLA-C locus belongs to the classical HLA-I gene, located between the HLA-A and B loci, encoding the HLA-C molecule, which has a gene length of about 3Kb, consisting of 7 introns and 8 exons. composition.
  • HLA-C has been found to be highly polymorphic like HLA-A and B molecules, and confirmed the importance of HLA-C molecules in clinical transplantation.
  • 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, to obtain HLA type-specific amplification products by PCR, and to determine the HLA type by electrophoresis analysis.
  • the principle of HLA-SSO is to design an HLA-specific oligonucleotide sequence as a probe, label the PCR product, and hybridize with the probe product (gene DNA to be detected). 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 generally can only reach the low-to-low level and cannot detect new alleles.
  • HLA-SBT based on Sanger sequencing is a method of PCR amplification
  • the DNA product is directly subjected to Sanger sequencing (capillary microelectrophoresis) to determine the nucleic acid sequence, thereby determining the HLA genotype high-resolution typing method, which has the characteristics of being intuitive, high-resolution and capable of detecting new alleles.
  • Sanger sequencing capillary microelectrophoresis
  • the HLA-SBT based on Sanger sequencing has complicated shortcomings, low throughput and high experimental cost, making 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.
  • 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 length of the PCR product should not exceed 700 bp, and the PCR primers of HLA-SBT based on the Sanger sequencing method are no longer applicable. Therefore, it is necessary to design a set of primers that are conservative and specific and have a PCR product length that meets the requirements of the new sequencing technology.
  • the present invention provides three pairs of PCR primers for amplifying the exons 2, 3 and 4 of the HLA-C gene, which are respectively Table 1. SEQ ID NOS: 1 and 2, 3 and 4, and 5 and 6 are shown.
  • the three pairs of PCR primers have good conservation and specificity, and can cover the full length sequences of exons 2, 3 and 4 of HLA-C sites, 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, 3 and 4 of HLA-C gene, characterized in that PCR amplification is carried out 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-C genotyping. Compared with the existing HLA-C genotyping method, since the method using the method of the present invention and the product of the amplification primer are controlled within 700 bp, it can be utilized based on further classification.
  • the present invention also provides a method of sequencing HLA-C gene 2, 3 and/or exon 4 in a sample, comprising the steps of:
  • the PCR product is purified;
  • the invention also provides a HLA-C genotyping method, the method comprising:
  • Illumina Solexa or Roche454.
  • the invention also provides a kit for performing HLA-C genotyping, the kit comprising a PCR amplification primer pair of the 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 a result of electrophoresis of a sample of HLA-C exon 2, 3, and 4 exon PCR products. From the electropherogram, the PCR product fragment size is a single band between 400 and 500 bp, wherein Lane M is a DNA standard molecular weight reference (DL 2000, Takara).
  • Figure 2 shows the DNA gel electrophoresis after HLA-Mix is interrupted, with a tapping area of 450-750 bp.
  • Lanes M are molecular weight standards (NEB-50bp DNA Ladder), lane 1 shows the gel map of HLA-Mix before tapping, and lane 2 shows the gel map of HLA-Mix after tapping.
  • Figure 3 shows a screenshot of the construct of the exon 2 consensus sequence for the HLA-C site of sample No. 2.
  • the sequencing sequence of the C site of the sample is aligned to the reference sequence by BWA software, and the consensus sequence of the exons 2, 2, and 4 of the sample C, respectively, is constructed, and then according to the SNP.
  • the linkage relationship is used to determine the haplotype sequence of each exon of the C site, and finally, the type of the sample is determined by the intersection of each exon haplotype sequence.
  • the 695-764 region of the gene sequence contains two heterozygous SNPs, and the SNP linkage relationship can be determined by readl and read2: AC, G- A ("" in the figure indicates the same base as the reference sequence).
  • the sequences correspond to the shaded portions of the C*010201 and C*07020101 type sequences, respectively.
  • the determination of the linkage relationship in other regions is similar.
  • Figure 4 shows the electropherograms of PCR products from 26 samples of HLA-C sites 2, 3 and 4.
  • Figure 5 shows the result of analysis of the sequencing peak of PCR product No. 1 template by uType software.
  • the result in the left result output column is C*08:01:01 C*15:05:01, and the template No.1 is known.
  • the same type The following embodiments are presented to enable a person of ordinary skill in the art to understand the present invention. The embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure.
  • the present invention employs the following method to design three pairs of PCR primers that amplify exons 2, 3 and 4 of HLA-C. All the latest HLA-C gene sequences were downloaded from the IMGT/HLA Internet site and saved to the local disk as an HLA-C data set; all the latest non-HLA-C HLA-I gene sequences were downloaded as a comparison data set. The two data sets were compared, and the conserved and specific sequences of each gene locus were searched at both ends and inside of exons 2, 3 and 4, and the designed PCR primer sequences were compared with the human whole genome sequence for homology. Since the HLA-C gene has high sequence similarity to other genes belonging to the HLA class I molecule, try to ensure primer 3 and terminal specificity when designing PCR primers to ensure primer amplification.
  • the specificity of the HLA-C gene is the specificity of the HLA-C 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.
  • Multiple pairs of candidate HLA-C primers meeting the design requirements were used to amplify a small number of template DNAs with common serotypes of HLA-C, and the most conservative and specific ones were screened for amplification of HLA- (: 3 pairs of PCR primers for exons 2, 3 and 4.
  • the method of the present invention can be any method of the present invention.
  • the method 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 sequence of tags is attached to the front end of the amplification primer to allow 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.
  • a unique primer label is introduced for each sample during the PCR process.
  • the detection process using the second generation DNA sequencing technology in addition to the PCR step must be processed one by one, other experiments can mix multiple samples and process at the same time, and finally the detection result of each sample can pass its unique primer label.
  • the sequence is retrieved.
  • the design of the primer label varies according to the experimental platform used.
  • the present invention mainly considers the following points when designing the primer label: 1: Avoid more than 3 in the primer label sequence (including 3 Single base repeat, 2: the total content of base A and base C in the same site of all primer tags is between 30% and 70% of the total base content, 3: GC of the primer tag sequence itself Content in
  • PCR-Free library adaptor refers to a designed base, the major cloning site, and the PCR-Free library linker can be directly ligated to both ends of the DNA fragment in the sequencing library by DNA ligase.
  • 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 link tags (adapter) Index )), constructing 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.
  • DNA was extracted from 95 blood samples of known HLA genotypes using the KingFisher automatic extractor.
  • the main steps are as follows: Take out the deep hole plate and one shallow hole plate of 6 Kingfisher automatic extractor, add a certain amount of matching reagents and mark according to the instructions, and place all the well plates with reagents as required. Location, selected program
  • PCR tag primers were made by synthesizing 5, PCR primers with different primer tags at the ends. These different PCR tag primers can be used for different samples.
  • the PCR primers are for exons 2, 3 and 4 of HLA-C. PCR 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.
  • each of the forward PCR primers has a forward primer label attached to a pair of primer tags at the 5' end, and a reverse primer label of a pair of primer tags attached to the 5' end of the reverse PCR primer.
  • the primer tag is added directly to the 5, end of the PCR primer when the primer is synthesized.
  • the 95 DNAs obtained in the sample extraction step were numbered 1-95 in sequence, and the PCR reaction was carried out in a 96-well plate.
  • the total number was 3 plates, numbered HLA-P-C2, HLA-P-C3, HLA-P-C4. (C2/3/4 indicates the amplified site), a negative control without template was set in the plate, and the primer used in the negative control was the same as the primer PI-96.
  • C3 96 ° C for 2 minutes; 95 ° C for 30 seconds 56 ° C for 30 seconds 72 ° C for 20 seconds (35 cycles); 15 ° C ⁇ ; C4: 96 ° C for 2 minutes; 95 ° C for 30 seconds 60 ° C for 30 seconds 72 ° C for 20 seconds (35 cycles); 15 ° C ⁇ .
  • the PCR reaction system of HLA-C is as follows:
  • PI nr CF 2/3/4 represents primer 5
  • PI nr CR 2/3/4 represents primer 5
  • each sample corresponds to a specific set of PCR primers.
  • Figure 1 shows the results of electrophoresis of the corresponding exon PCR products of the first 20 samples of HLA-C.
  • the DNA molecule is labeled DL 2000 ( Takara).
  • the gel image has a series of fragments ranging from 400 bp to 500 bp.
  • HLA-C2-Mix Mix 20 ⁇ in a 3 ml tube, labeled HLA-C2-Mix, and perform the same operation on the other 2 96-well plates, labeled HLA-C3-Mix and HLA-C4-Mix, shake and mix 200 ⁇ L from HLA-C2-Mix, HLA-C3-Mix and HLA-C4-Mix in a 1.5 ml tube, labeled HLA-Mix, 500 ⁇ DNA mixture from HLA-Mix Purified by a Qiagen DNA Purification kit (QIAGE) (specific purification steps are detailed in the instructions), purified 200 ⁇ l ⁇ , and the HLA-Mix DNA concentration was determined to be 50 ng 1 by Nanodrop 8000 (Thermo Fisher Scientific).
  • QIAGE Qiagen DNA Purification kit
  • DNA end-repair reaction of the purified product the system is as follows (reagents are purchased from
  • reaction conditions were as follows: at 20 ° C, in a Thermomixer (Eppendorf) bath for 30 minutes.
  • reaction product was purified by QIAqukk PCR Purification Kit and dissolved in 32 ⁇ M EB (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):
  • reaction product was purified by MiniElute PCR Purification Kit (QIAGE) and dissolved in a 38 ⁇ M 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 determined by real-time PCR (QPCR) as follows:
  • HLA-Mix 30 ⁇ l of HLA-Mix was recovered using 2% low melting point agarose gel. The electrophoresis conditions were 100V for 100 minutes.
  • the DNA standard molecular weight reference is a 50 bp DNA Ladder from NEB.
  • the gel was recovered to recover DNA fragments ranging from 400-750 bp in length (Fig. 2).
  • the gel recovery product was recovered and purified by QIAquick PGR Purification Kit (QIAGEN). After purification, the volume was 32 ⁇ , and the DNA concentration was 17.16 nM by real-time PCR (QPCR).
  • the sequencing result of Illumina GA is a series of DNA sequences, sequenced by search The positive and negative primer tag sequences and primer sequences in the results, and the corresponding samples of each primer tag are established.
  • the (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-C.
  • the screenshot of Figure 3 exemplifies the process of constructing the exon 2 consensus sequence for the HLA-C site of sample 1.
  • the DNA sequence of the sequenced HLA-C intron is aligned with the sequence database of the corresponding exons of HLA-C in the IMGT HLA professional database, and the HLA-C gene of the corresponding sample is matched by 100% of the sequence alignment results.
  • Type For all 95 samples, the obtained classification results were completely consistent with the original known classification results.
  • the specific results of the samples No. 1-32 were compared with the original classification results of the samples as follows: (As shown in Table 3, all the test results are the same as the original The test results are the same).
  • C-F2, C-R2, C-F3, C-R3, C-F4, and C-R4 were amplified by single-tube PCR, and the PCR procedures of each pair of primers were as follows:
  • the PCR reaction system of HLA-C is as follows:
  • the PCR product was detected by agarose gel electrophoresis (Fig. 4) 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 ⁇ l of ultrapure water to the wells to be used, attach the remaining membrane to the remaining pores, let stand for 15 minutes at room temperature or connect to the pumping On the filter system, take -10 Pa for 5 minutes. Each time the purification plate is removed from the suction 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 observed under illumination, not complete. The liquid surface reflects the luster.
  • the sequencing reaction was carried out by using the above purified PCR product as a template, and the conditions for the sequencing reaction were: 96 ° C for 2 minutes; 96 ° C for 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 at 3000 g for 1 minute. In a 96-well plate, add 2 ⁇ 0.125 mol/L EDTA-Na2 solution, 33 ⁇ 85% ethanol per 5 ⁇ reaction solution, cover with a silica gel pad, and shake well for 3 minutes at 4 °C. Centrifuge at 3000 g for 30 minutes. 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.
  • a 96-well plate was added with 50 ⁇ 70% ethanol per well, covered with a silica gel 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 to an ethanol free odor.
  • 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).
  • Wegner KM Massive parallel MHC genotyping: titanium that shines. Molecular Ecology 2009, 18: 1818-1820.

Abstract

A method for amplifying exons 2, 3 and 4 of HLA-C gene and the specific primer pairs used in the method are provided. A method for HLA-C genotyping and the amplification primer pairs used in the method are also provided.

Description

HLA-C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-C 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 ( human leucocyte antigen, HLA )是人类免疫系 统的重要组成部分， 也是目前所知人体最复杂的遗传多态性系统。 根 据其结构、 组织分布和功能等方面的特点， HLA分子可分为 HLA- I 、 HLA- II和 HLA-m等三类分子， 其中 HLA- I类分子和 HLA- II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； 临床移植研究发现， 移植 物的长期存活率与供者和受者双方的 HLA- I和 HLA- II分子的匹配程 度呈正相关性， 当供受双方的 HLA分子的匹配程度越高， 移植物的长 期存活率就越高。  FIELD OF THE INVENTION The present invention relates to the field of molecular biology, and in particular to a method for HLA-C genotyping, and specific primers used in the method . BACKGROUND OF THE INVENTION Human leucocyte antigen (HLA) is an important component of the human immune system and is the most complex genetic polymorphism system known to the human body. According to its structure, tissue distribution and function, HLA molecules can be divided into three types of molecules: HLA-I, HLA-II and HLA-m. Among them, HLA-I and HLA-II molecular functions are mainly immune. Rejection-related, HLA-m class molecular function is mainly related to the synthesis of immune-related partial complement system and inflammation-related factors; clinical transplantation studies found that graft long-term survival rate and HLA-I and HLA of both donors and recipients - The degree of matching of the II molecules is positively correlated. The higher the degree of matching between the donor and recipient HLA molecules, the higher the long-term survival rate of the graft.
其中 HLA-C位点属于经典的 HLA- I类基因，位于 HLA-A 、 B 位点之间， 编码 HLA-C分子， 其基因长度约 3Kb, 由 7个内含子和 8 个外显子组成。 近年来， 随着 HLA 基因分型技术的不断发展， 人们 逐渐发现 HLA-C和 HLA-A、 B分子一样， 具有高度多态性， 并证实 了 HLA-C分子在临床移植中的重要性。  The HLA-C locus belongs to the classical HLA-I gene, located between the HLA-A and B loci, encoding the HLA-C molecule, which has a gene length of about 3Kb, consisting of 7 introns and 8 exons. composition. In recent years, with the continuous development of HLA genotyping technology, HLA-C has been found to be highly polymorphic like HLA-A and B molecules, and confirmed the importance of HLA-C molecules in clinical transplantation.
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）。  Current international standard HLA typing techniques include PCR-SSP (sequence-specific primer polymerase chain reaction), PCR-SSO (polymerase chain reaction oligonucleotide probe hybridization) and PCR-SBT (polymerase chain reaction) The product was directly sequenced and classified).
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， 分辨率一般只能到达中低水平且都不能检 测新的等位基因。  The principle of HLA-SSP is to design a set of allele-specific primers, to obtain HLA type-specific amplification products by PCR, and to determine the HLA type by electrophoresis analysis. The principle of HLA-SSO is to design an HLA-specific oligonucleotide sequence as a probe, label the PCR product, and hybridize with the probe product (gene DNA to be detected). 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 generally can only reach the low-to-low level and cannot detect new alleles.
基于 Sanger法测序的 HLA-SBT是一种通过对 PCR扩增后的 DNA产物直接进行 Sanger法测序（毛细管微电泳），测定核酸序列， 从而判断 HLA基因型别的高分辨分型方法， 其具有直观、 高分辨且 能检测新的等位基因的特点。 但基于 Sanger法测序的 HLA-SBT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。 HLA-SBT based on Sanger sequencing is a method of PCR amplification The DNA product is directly subjected to Sanger sequencing (capillary microelectrophoresis) to determine the nucleic acid sequence, thereby determining the HLA genotype high-resolution typing method, which has the characteristics of being intuitive, high-resolution and capable of detecting new alleles. However, the HLA-SBT based on Sanger sequencing has complicated shortcomings, low throughput and high experimental cost, making it difficult to apply to large-scale HLA high-resolution typing projects.
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、 实验流程简单、 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。  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 technology (sequencing technology based on the Sanger sequencing principle), 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.
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。  Due to the characteristics of the new sequencing technology, the length of the PCR product should not exceed 700 bp, and the PCR primers of HLA-SBT based on the Sanger sequencing method are no longer applicable. Therefore, it is necessary to design a set of primers that are conservative and specific and have a PCR product length that meets the requirements of the new sequencing technology.
参考文献  references
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[6】 Elaine R. Mardis. The impact of next-generation sequencin technology on genetics. Trends in Genetics.2008,24: 133-141.  [6] Elaine R. Mardis. The impact of next-generation sequencin technology on genetics. Trends in Genetics. 2008, 24: 133-141.
[7] Hoffmann C， Minkah N, Leipzig J， Wang G, Arens MQ， Tebas P， Bushman FD. DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations. Nucleic Acids Res. 2007;35(13):e91. 发明内容 为了将第二代测序技术用于 HLA-C基因分型，本发明提供了用于 扩增 HLA-C基因的 2、 3和 4号外显子的 3对 PCR引物， 它们分别是 表 1中显示的 SEQ ID NO: 1和 2、 3和 4以及 5和 6。 所述 3对 PCR 引物具有良好的保守性和特异性， 并且可覆盖 HLA-C位点 2、 3和 4 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。  [7] Hoffmann C, Minkah N, Leipzig J, Wang G, Arens MQ, Tebas P, Bushman FD. DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations. Nucleic Acids Res. 2007;35(13):e91. SUMMARY OF THE INVENTION In order to use the second generation sequencing technology for HLA-C genotyping, the present invention provides three pairs of PCR primers for amplifying the exons 2, 3 and 4 of the HLA-C gene, which are respectively Table 1. SEQ ID NOS: 1 and 2, 3 and 4, and 5 and 6 are shown. The three pairs of PCR primers have good conservation and specificity, and can cover the full length sequences of exons 2, 3 and 4 of HLA-C sites, and the PCR products are less than 700 bp in length, which meets the requirements of normal Illumina Solexa sequencing. In addition, the primers of the present invention are also suitable for Sanger sequencing.
表 1. HLA-C基因 2、 3和 4号外显子的 PCR引物:  Table 1. PCR primers for HLA-C gene exons 2, 3 and 4:
Figure imgf000004_0001
Figure imgf000004_0001
本发明还提供了一种新的 HLA-C基因 2、 3和 4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。  The present invention also provides a novel method for amplifying exon 2, 3 and 4 of HLA-C gene, characterized in that PCR amplification is carried out using the amplification primer pair of the present invention, and the sequence of the amplification primer pair is shown. In Table 1.
由于能够通过 PCR反应扩增出 HLA-C的 2、 3和 4号外显子， 因 此， 本发明的方法特别有利地可用于进行 HLA-C基因分型。 与现有的 HLA-C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 700 bp以内， 因此在进一步进行分型时， 可以利用基于  Since the exons 2, 3 and 4 of HLA-C can be amplified by a PCR reaction, the method of the present invention is particularly advantageously used for HLA-C genotyping. Compared with the existing HLA-C genotyping method, since the method using the method of the present invention and the product of the amplification primer are controlled within 700 bp, it can be utilized based on further classification.
Illumina Solexa测序技术的 HLA-SBT。  HLA-SBT for Illumina Solexa sequencing technology.
本发明还提供了一种对样品中的 HLA-C基因 2、 3和 /或 4号外显 子进行测序的方法， 其包括步骤：  The present invention also provides a method of sequencing HLA-C gene 2, 3 and/or exon 4 in a sample, comprising the steps of:
1 ) 提供一个样品并提取该样品的 DNA;  1) providing a sample and extracting DNA of the sample;
2 ) 将本发明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； 2) using a primer pair of the present invention for amplifying the DNA to obtain a PCR product, Preferably, the PCR product is purified;
3 ) 对所述 PCR产物进行测序， 所述测序优选是通过第二代测 序法， 所述第二代测序方法是例如 Illumina Solexa或 Roche454。  3) sequencing the PCR product, preferably by a second generation sequencing method, such as Illumina Solexa or Roche 454.
本发明还提供了一种 HLA-C基因分型方法， 所述方法包括：  The invention also provides a HLA-C genotyping method, the method comprising:
1) 使用本发明的引物对进行 PCR扩增， 扩增待测样本的 HLA-C 基因 2、 3和 /或 4号外显子；  1) PCR amplification using the primer pair of the present invention to amplify the HLA-C gene 2, 3 and/or exon 4 of the sample to be tested;
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， 其中所述测序是通过 Sanger 测序法， 或者是通过第二代测序法， 所述第二代测序方法是例如  2) Sequencing the amplified exons and comparing the sequencing results to standard sequences in the database to determine genotyping results, either by Sanger sequencing or by second generation sequencing Method, the second generation sequencing method is for example
Illumina Solexa或 Roche454。 Illumina Solexa or Roche454.
另一方面， 本发明还提供了一种用于进行 HLA-C基因分型的试剂 盒， 所述试剂盒中包括本发明的 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。  In another aspect, the invention also provides a kit for performing HLA-C genotyping, the kit comprising a PCR amplification primer pair of the invention. In one embodiment, the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.
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  The amplification primer pair and the genotyping method provided by the present invention can be amplified
HLA-C的 2、 3和 4号外显子的基础上进行基因分型。 因此相对于现有 技术而言， 该分型利用了 Illumina Solexa测序技术， 提高了通量， 简 化了流程， 同时还节省了时间和成本。 附图说明 图 1为部分样本的 HLA-C 2，3，4号外显子 PCR产物电泳结果，从电 泳图上看， PCR产物片段大小分别为位于 400-500 bp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）。  Genotyping was performed on the basis of exons 2, 3 and 4 of HLA-C. Therefore, compared to the prior art, this type of classification utilizes Illumina Solexa sequencing technology to increase throughput and streamline the process while saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a result of electrophoresis of a sample of HLA-C exon 2, 3, and 4 exon PCR products. From the electropherogram, the PCR product fragment size is a single band between 400 and 500 bp, wherein Lane M is a DNA standard molecular weight reference (DL 2000, Takara).
图 2显示对 HLA-Mix打断后 DNA电泳凝胶割胶的情况，割胶区 域为 450-750 bp区域。其中泳道 M是分子量标准物（ NEB-50bp DNA Ladder ) , 泳道 1示出割胶前 HLA-Mix的胶图， 泳道 2示出割胶后 HLA-Mix的胶图。  Figure 2 shows the DNA gel electrophoresis after HLA-Mix is interrupted, with a tapping area of 450-750 bp. Lanes M are molecular weight standards (NEB-50bp DNA Ladder), lane 1 shows the gel map of HLA-Mix before tapping, and lane 2 shows the gel map of HLA-Mix after tapping.
图 3显示 2号样品的 HLA-C位点的 2号外显子一致性序列构建程序 截图。 首先， 该样本的 C位点的测序序列通过 BWA软件比对到参考序列 上， 分别构建出该样本 C位点 2、 2、 4号外显子的一致性序列， 再才艮据 SNP之间的连锁关系来确定 C位点各外显子的单体型序列， 最后， 由各 外显子单体型序列的交集确定该样本的型别。 如图所示， 在 2 号样本 C 基因序列 695 - 764区域含有 2个杂合 SNP，由 readl和 read2可确定 SNP 连锁关系分别为： A-C，G- A(图中的"… "表示与参考序列相同的碱基）。 其序列分别对应 C*010201和 C*07020101型别序列的阴影部分。 其 他区域的连锁关系的判定与此类似。 Figure 3 shows a screenshot of the construct of the exon 2 consensus sequence for the HLA-C site of sample No. 2. First, the sequencing sequence of the C site of the sample is aligned to the reference sequence by BWA software, and the consensus sequence of the exons 2, 2, and 4 of the sample C, respectively, is constructed, and then according to the SNP. The linkage relationship is used to determine the haplotype sequence of each exon of the C site, and finally, the type of the sample is determined by the intersection of each exon haplotype sequence. As shown in the sample No. 2 The 695-764 region of the gene sequence contains two heterozygous SNPs, and the SNP linkage relationship can be determined by readl and read2: AC, G- A ("" in the figure indicates the same base as the reference sequence). The sequences correspond to the shaded portions of the C*010201 and C*07020101 type sequences, respectively. The determination of the linkage relationship in other regions is similar.
图 4显示 26份样本 HLA-C位点 2、 3和 4号外显子 PCR产物电泳图。  Figure 4 shows the electropherograms of PCR products from 26 samples of HLA-C sites 2, 3 and 4.
26份样本 HLA-C位点 2、 3和 4号外显子 PCR产物电泳图， 如图中所示， 所有 PCR产物均小于 500 bp, 且电泳条带单一， 无明显的非特异性条带， 同一对引物对于各样本的扩增效率均一。  26 samples of HLA-C site 2, 3 and 4 exon PCR product electropherogram, as shown in the figure, all PCR products are less than 500 bp, and the electrophoresis bands are single, no obvious non-specific bands, the same The amplification efficiency of the primers for each sample was uniform.
图 5显示扩增 1号模板 PCR产物测序峰图经 uType软件分析的结果， 左侧结果输出栏中显示结果 C*08:01:01 C*15:05:01，与 1号模板原已知型别 相同。 具体实施方式 提出以下实施例， 以便使本领域普通技术人员更好地理解本发明， 所述实施例仅出于示例性目的， 并非意在限制本公开的范围。  Figure 5 shows the result of analysis of the sequencing peak of PCR product No. 1 template by uType software. The result in the left result output column is C*08:01:01 C*15:05:01, and the template No.1 is known. The same type. The following embodiments are presented to enable a person of ordinary skill in the art to understand the present invention. The embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure.
本发明采用如下方法设计扩增 HLA-C的 2、 3和 4号外显子的 3 对 PCR引物。从 IMGT/HLA因特网站点下载所有最新 HLA-C基因序 列， 然后保存到本地磁盘中作为 HLA-C数据集； 同时下载所有最新非 HLA-C的 HLA-I类基因序列作为比较数据集。 将两数据集进行比较， 在 2、 3和 4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。 由于 HLA-C基因与同属于 HLA-I类分子的其它基因具有很高的序列相似 性， 在设计 PCR引物时尽量保证引物 3，末端特异， 确保引物扩增  The present invention employs the following method to design three pairs of PCR primers that amplify exons 2, 3 and 4 of HLA-C. All the latest HLA-C gene sequences were downloaded from the IMGT/HLA Internet site and saved to the local disk as an HLA-C data set; all the latest non-HLA-C HLA-I gene sequences were downloaded as a comparison data set. The two data sets were compared, and the conserved and specific sequences of each gene locus were searched at both ends and inside of exons 2, 3 and 4, and the designed PCR primer sequences were compared with the human whole genome sequence for homology. Since the HLA-C gene has high sequence similarity to other genes belonging to the HLA class I molecule, try to ensure primer 3 and terminal specificity when designing PCR primers to ensure primer amplification.
HLA-C基因的特异性。 同时使 PCR产物的长度小于 700 bp , 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-C引 物用于扩增少数具有 HLA-C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 HLA- (：的 2、 3和 4号外显子的 3对 PCR引物。 The specificity of the HLA-C gene. At the same time, 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. Multiple pairs of candidate HLA-C primers meeting the design requirements were used to amplify a small number of template DNAs with common serotypes of HLA-C, and the most conservative and specific ones were screened for amplification of HLA- (: 3 pairs of PCR primers for exons 2, 3 and 4.
本发明 述方法可以
Figure imgf000006_0001
The method of the present invention can
Figure imgf000006_0001
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。 The method 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. ' For example, in the sequencing process using the second-generation sequencing technology, 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.
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  A sequence of tags is attached to the front end of the amplification primer to allow 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 PCR-index/barcode technology, and a unique primer label is introduced for each sample during 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 experiments can mix multiple samples and process at the same time, and finally the detection result of each sample can pass its unique primer label. The sequence is retrieved. The design of the primer label varies according to the experimental platform used. 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 primer label sequence (including 3 Single base repeat, 2: the total content of base A and base C in the same site of all primer tags is between 30% and 70% of the total base content, 3: GC of the primer tag sequence itself Content in
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。 Between 40-60%, 4: Sequence difference between primer tags is greater than 4 bases, 5: Primer tag sequences avoid sequences with high similarity to Illumina GA sequencing primers, 6: Reduce primer tag sequences added to PCR After the primers, the hairpin and the dimer caused by the PCR primers appeared.
术语" PCR-Free文库接头（ adapter ) "是指经设计的一段碱基， 其主 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。  The term "PCR-Free library adaptor" refers to a designed base, the major cloning site, and the PCR-Free library linker can be directly ligated to both ends of the DNA fragment in the sequencing library by DNA ligase. 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 link tags (adapter) Index )), constructing 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. For example, a PCR-FREE library linker is used in the present invention from ILLUMIA.
在如下的实施例中，用筛选出的 3对 PCR引物，对 95例已知 HLA 常见基因型别的血样进行 HLA-C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-C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 Il miina GA进行 HLA-C基因分型 1. 样本 DNA提取 In the following examples, 95 pairs of known HLA common genotypes were subjected to HLA-C site PCR amplification using the selected 3 pairs of PCR primers, and the amplified products were subjected to Sanger method and second generation sequencing method. Sequencing was performed. The sequencing results were used for HLA-C typing and the conservatism and specificity of the PCR primers were verified by comparison with the original typing results. Example 1. HLA-C genotyping using the second generation sequencing technology Il miina GA 1. Sample DNA extraction
使用 KingFisher自动提取仪提取 95例已知 HLA基因型别的血样 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，依照说明书分别加入一定量配套的试剂并标记， 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  DNA was extracted from 95 blood samples of known HLA genotypes using the KingFisher automatic extractor. The main steps are as follows: Take out the deep hole plate and one shallow hole plate of 6 Kingfisher automatic extractor, add a certain amount of matching reagents and mark according to the instructions, and place all the well plates with reagents as required. Location, selected program
"Bioeasy— 200μ1 Blood DNA— KF.msz"程序， 按下" start"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, 作为下一步 PCR中的模板。 "Bioeasy - 200μ1 Blood DNA - KF.msz" program, press "start" to execute the program for nucleic acid extraction. At the end of the program, 100 μΐ of the eluted product in the plate Elution was collected as the extracted DNA as a template for the next PCR.
2. PCR扩增  2. PCR amplification
通过合成 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，这些不同的 PCR标签引物可以用于不同的样本，所述 PCR 引物是针对 HLA-C的 2、 3和 4号外显子 PCR引物。 其后通过 PCR 反应在 PCR产物两端引入引物标签，从而特异性标记来自不同样本的 PCR产物。  Different PCR tag primers were made by synthesizing 5, PCR primers with different primer tags at the ends. These different PCR tag primers can be used for different samples. The PCR primers are for exons 2, 3 and 4 of HLA-C. PCR 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.
以 95套 PCR标签引物来分别扩增 95份 DNA样本，每套 PCR标 签引物由用于扩增 HLA-C的 2、 3和 4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。  95 sets of PCR-labeled primers were used to amplify 95 DNA samples, each set of PCR-label primers from PCR primers for amplification of HLA-C exons 2, 3 and 4 (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 5' end, and a reverse primer label of a pair of primer tags attached to the 5' end of the reverse PCR primer. The primer tag is added directly to the 5, end of the PCR primer when the primer is synthesized.
把样本提取步骤中所得的 95份 DNA, 依次编号 1-95 , PCR反应 在 96孔板中进行， 共 3板， 编号分别为 HLA-P-C2、 HLA-P-C3 , HLA-P-C4 ( C2/3/4表示扩增的位点）， 板内设置一个不添加模板的阴 性对照， 阴性对照所用引物与引物 PI-96相同。 实验的同时， 记录下 每对引物标签对应的样本编号信息。  The 95 DNAs obtained in the sample extraction step were numbered 1-95 in sequence, and the PCR reaction was carried out in a 96-well plate. The total number was 3 plates, numbered HLA-P-C2, HLA-P-C3, HLA-P-C4. (C2/3/4 indicates the amplified site), a negative control without template was set in the plate, and the primer used in the negative control was the same as the primer PI-96. At the same time as the experiment, record the sample number information corresponding to each pair of primer labels.
引物标签的相关信息:  Primer label related information:
编号 正向 反向 96孔板位置 对应模板 No. Forward Reverse 96-well plate position Corresponding template
PI-1 TCGCAGACATCA TGACACGATGCT A01 1 PI-1 TCGCAGACATCA TGACACGATGCT A01 1
PI-2 TACATCGCACTA TACAGATGCTGA A02 2  PI-2 TACATCGCACTA TACAGATGCTGA A02 2
PI-3 CTCGATGAGTAC ACGTCTAGACAC A03 3  PI-3 CTCGATGAGTAC ACGTCTAGACAC A03 3
PI-4 TCTGTATACTCA TGCTGTAGTGAC A04 4  PI-4 TCTGTATACTCA TGCTGTAGTGAC A04 4
PI-5 TATCTGCTCATA AGATATCGAGCT A05 5  PI-5 TATCTGCTCATA AGATATCGAGCT A05 5
PI-6 TACATGCTGAGC ACGTGTCTATCA A06 6  PI-6 TACATGCTGAGC ACGTGTCTATCA A06 6
PI-7 TCATATCGCGAT AGATCGTATAGC A07 7  PI-7 TCATATCGCGAT AGATCGTATAGC A07 7
PI-8 ACAGATGCACGC ATCTCGTGACAG A08 8 PI-9 TAGATCGTACAT ACTAGTACACGC A09 9PI-8 ACAGATGCACGC ATCTCGTGACAG A08 8 PI-9 TAGATCGTACAT ACTAGTACACGC A09 9
PI-10 ACTACACGTCTC ATAGTCACGCGT A10 10PI-10 ACTACACGTCTC ATAGTCACGCGT A10 10
PI-11 AGACTCGCGTAT TACTAGCTGACG All 11PI-11 AGACTCGCGTAT TACTAGCTGACG All 11
PI-12 ATACTAGTGCTC TGTATCGTGCTC A12 12PI-12 ATACTAGTGCTC TGTATCGTGCTC A12 12
PI-13 CACGATGACATC TAGTGAGCGCAC B01 13PI-13 CACGATGACATC TAGTGAGCGCAC B01 13
PI-14 TGCTGTCTCGAG CATAGCAGTGTC B02 14PI-14 TGCTGTCTCGAG CATAGCAGTGTC B02 14
PI-15 TGTGCTCGAGTC TCTGATCGAGCA B03 15PI-15 TGTGCTCGAGTC TCTGATCGAGCA B03 15
PI-16 CACTCGTACATC AGCGATGCTCAT B04 16PI-16 CACTCGTACATC AGCGATGCTCAT B04 16
PI-17 CGACGTGCTCGC CGCGTACTGCAG B05 17PI-17 CGACGTGCTCGC CGCGTACTGCAG B05 17
PI-18 ACGCATCTATAC CTAGTATCGCAG B06 18PI-18 ACGCATCTATAC CTAGTATCGCAG B06 18
PI-19 CGAGATGACTCT TGTATACACGAT B07 19PI-19 CGAGATGACTCT TGTATACACGAT B07 19
PI-20 ACTGTCTCGAGC ACGTAGCGCACA B08 20PI-20 ACTGTCTCGAGC ACGTAGCGCACA B08 20
PI-21 CATCTGCTATAG TCTAGCTCATGA B09 21PI-21 CATCTGCTATAG TCTAGCTCATGA B09 21
PI-22 ACGCACTCTAGA CTATGCACTGAT BIO 22PI-22 ACGCACTCTAGA CTATGCACTGAT BIO 22
PI-23 TGAGATACAGTA ATCTGCTATGAC Bll 23PI-23 TGAGATACAGTA ATCTGCTATGAC Bll 23
PI-24 ACTCATCGTGCT TAGAGCTGTCAC B12 24PI-24 ACTCATCGTGCT TAGAGCTGTCAC B12 24
PI-25 TACACTGTCTAT CAGCACATAGAT C01 25PI-25 TACACTGTCTAT CAGCACATAGAT C01 25
PI-26 CACAGTACTCGC CTGCTAGTGTAT C02 26PI-26 CACAGTACTCGC CTGCTAGTGTAT C02 26
PI-27 TGTACTATCATA TGTGATAGACAC C03 27PI-27 TGTACTATCATA TGTGATAGACAC C03 27
PI-28 CTAGTACTGACG AGCGAGTCTACT C04 28PI-28 CTAGTACTGACG AGCGAGTCTACT C04 28
PI-29 TAGACTGAGCTA ACATACTGAGAC C05 29PI-29 TAGACTGAGCTA ACATACTGAGAC C05 29
PI-30 CAGACGCGTGAG TACATCTCGTAT C06 30PI-30 CAGACGCGTGAG TACATCTCGTAT C06 30
PI-31 CGCGACATCACG TAGCGATGAGAC C07 31PI-31 CGCGACATCACG TAGCGATGAGAC C07 31
PI-32 ACACTCATAGAT CTATCATGACAC C08 32PI-32 ACACTCATAGAT CTATCATGACAC C08 32
PI-33 AGCGTATACTAG CATACTCACGTA C09 33PI-33 AGCGTATACTAG CATACTCACGTA C09 33
PI-34 TGTCGTGCTATC ACATGACTCACG CIO 34PI-34 TGTCGTGCTATC ACATGACTCACG CIO 34
PI-35 CGCTAGACTGTA TACTATAGTCGA Cll 35PI-35 CGCTAGACTGTA TACTATAGTCGA Cll 35
PI-36 ACAGTGTAGCGC TGATATGCTACA C12 36PI-36 ACAGTGTAGCGC TGATATGCTACA C12 36
PI-37 CACTCTATCGAC TCACGCGATGAG D01 37PI-37 CACTCTATCGAC TCACGCGATGAG D01 37
PI-38 ACACTCTAGTCA ACGTAGATCTAT D02 38PI-38 ACACTCTAGTCA ACGTAGATCTAT D02 38
PI-39 CATATGAGATCG AGCAGAGTGCTC D03 39PI-39 CATATGAGATCG AGCAGAGTGCTC D03 39
PI-40 CAGCTATCATAC CACTGCAGACGA D04 40PI-40 CAGCTATCATAC CACTGCAGACGA D04 40
PI-41 TATACTCTAGAT TGCATAGAGCGC D05 41PI-41 TATACTCTAGAT TGCATAGAGCGC D05 41
PI-42 TGTATGCTCGTC TCGTGACAGATC D06 42PI-42 TGTATGCTCGTC TCGTGACAGATC D06 42
PI-43 TAGTGATGCTCT ACGAGCTGATAT D07 43PI-43 TAGTGATGCTCT ACGAGCTGATAT D07 43
PI-44 AGACTCTGAGTC CTGATAGTATCA D08 44PI-44 AGACTCTGAGTC CTGATAGTATCA D08 44
PI-45 CTCATAGACTAC ATCGCGAGTGAC D09 45PI-45 CTCATAGACTAC ATCGCGAGTGAC D09 45
PI-46 TCGCTCACTACA TGTCTCGACATC D10 46PI-46 TCGCTCACTACA TGTCTCGACATC D10 46
PI-47 ATAGAGTCTCAT CGCATAGCGTAT Dll 47PI-47 ATAGAGTCTCAT CGCATAGCGTAT Dll 47
PI-48 CGAGACACTCGC TCGTAGTCTACA D12 48PI-48 CGAGACACTCGC TCGTAGTCTACA D12 48
PI-49 CAGCATACTATC TCGTGATACAGA E01 49PI-49 CAGCATACTATC TCGTGATACAGA E01 49
PI-50 CAGCTATAGTCT ATGCAGATATCT E02 50PI-50 CAGCTATAGTCT ATGCAGATATCT E02 50
PI-51 TCTATCGATGCA ACACGCAGATCG E03 51PI-51 TCTATCGATGCA ACACGCAGATCG E03 51
PI-52 CATGAGTATAGC CTAGCTGACGTA E04 52PI-52 CATGAGTATAGC CTAGCTGACGTA E04 52
PI-53 TAGCATATCGAG TACACGTATGAG E05 53PI-53 TAGCATATCGAG TACACGTATGAG E05 53
PI-54 ACGACTCGCTAC TCATGACTAGTA E06 54PI-54 ACGACTCGCTAC TCATGACTAGTA E06 54
PI-55 TAGCATACACGC TGACGCGTATAC E07 55PI-55 TAGCATACACGC TGACGCGTATAC E07 55
PI-56 CGTCATATGCAG TATAGCGATGAC E08 56PI-56 CGTCATATGCAG TATAGCGATGAC E08 56
PI-57 TGCAGCGAGTAC TCGACGCTAGCG E09 57 PI-58 CGTGTCGACAGA CAGTCGTGAGCA E10 58PI-57 TGCAGCGAGTAC TCGACGCTAGCG E09 57 PI-58 CGTGTCGACAGA CAGTCGTGAGCA E10 58
PI-59 ACTCGACGTGAG ACGCGAGTGATA Ell 59 PI-59 ACTCGACGTGAG ACGCGAGTGATA Ell 59
PI-60 ACTCGTCTGACG TGCTATCACTGA E12 60  PI-60 ACTCGTCTGACG TGCTATCACTGA E12 60
PI-61 CATACTGTATCT TACATAGATGTC F01 61  PI-61 CATACTGTATCT TACATAGATGTC F01 61
PI-62 TCTACTCGTGAC CACGTATAGTGA F02 62  PI-62 TCTACTCGTGAC CACGTATAGTGA F02 62
PI-63 CTGCACTAGACA ACTCATATCGCA F03 63  PI-63 CTGCACTAGACA ACTCATATCGCA F03 63
PI-64 ACACGAGCTCAT CACTCATATCGA F04 64  PI-64 ACACGAGCTCAT CACTCATATCGA F04 64
PI-65 TACAGATAGTCT TCGTCTGTGATA F05 65  PI-65 TACAGATAGTCT TCGTCTGTGATA F05 65
PI-66 TACACTCGTGCT TGACGCTCATCT Γ06 66  PI-66 TACACTCGTGCT TGACGCTCATCT Γ06 66
PI-67 TACATGTGACGA TCGTACATGCTC F07 67  PI-67 TACATGTGACGA TCGTACATGCTC F07 67
PI-68 TGTATGATCTCG CACTGTGCTCAT F08 68  PI-68 TGTATGATCTCG CACTGTGCTCAT F08 68
PI-69 CAGTACACTCTA ACTGCATGATCG F09 69  PI-69 CAGTACACTCTA ACTGCATGATCG F09 69
PI-70 CATACTATCACG TCGTGTCACTAC F10 70  PI-70 CATACTATCACG TCGTGTCACTAC F10 70
PI-71 CACTATACAGAT CGACACGTACTA Fll 71  PI-71 CACTATACAGAT CGACACGTACTA Fll 71
PI-72 ATATCGTAGCAT TCGTGATCACTA F12 72  PI-72 ATATCGTAGCAT TCGTGATCACTA F12 72
PI-73 TAGTCTATACAT AGACGCTGTCGA G01 73  PI-73 TAGTCTATACAT AGACGCTGTCGA G01 73
PI-74 TGTCACAGTGAC TCATATGATCGA G02 74  PI-74 TGTCACAGTGAC TCATATGATCGA G02 74
PI-75 ATCGACTATGCT CGATCATATGAG G03 75  PI-75 ATCGACTATGCT CGATCATATGAG G03 75
PI-76 ATACTAGCATCA TCATGCTGACGA G04 76  PI-76 ATACTAGCATCA TCATGCTGACGA G04 76
PI-77 CACTGACGCTCA CACTACATCGCT G05 77  PI-77 CACTGACGCTCA CACTACATCGCT G05 77
PI-78 TCGCTCATCTAT TAGTACAGAGCT G06 78  PI-78 TCGCTCATCTAT TAGTACAGAGCT G06 78
PI-79 TGTATCACGAGC ATGATCGTATAC G07 79  PI-79 TGTATCACGAGC ATGATCGTATAC G07 79
PI-80 TACTGCTATCTC CGCTGCATAGCG G08 80  PI-80 TACTGCTATCTC CGCTGCATAGCG G08 80
PI-81 CGCGAGCTCGTC ACTCGATGAGCT G09 81  PI-81 CGCGAGCTCGTC ACTCGATGAGCT G09 81
PI-82 TAGAGTCTGTAT TGTCTATCACAT G10 82  PI-82 TAGAGTCTGTAT TGTCTATCACAT G10 82
PI-83 TACTATCGCTCT TATGTGACATAC Gil 83  PI-83 TACTATCGCTCT TATGTGACATAC Gil 83
PI-84 TAGATGACGCTC TACTCGTAGCGC G12 84  PI-84 TAGATGACGCTC TACTCGTAGCGC G12 84
PI-85 TCGCGTGACATC ATCTACTGACGT HOI 85  PI-85 TCGCGTGACATC ATCTACTGACGT HOI 85
PI-86 ACACGCTCTACT ACAGTAGCGCAC H02 86  PI-86 ACACGCTCTACT ACAGTAGCGCAC H02 86
PI-87 TACATAGTCTCG CTAGTATCATGA H03 87  PI-87 TACATAGTCTCG CTAGTATCATGA H03 87
PI-88 TGAGTAGCACGC TCGATCATGCAG H04 88  PI-88 TGAGTAGCACGC TCGATCATGCAG H04 88
PI-89 TAGATGCTATAC TACATGCACTCA H05 89  PI-89 TAGATGCTATAC TACATGCACTCA H05 89
PI-90 ATCGATGTCACG CAGCTCGACTAC H06 90  PI-90 ATCGATGTCACG CAGCTCGACTAC H06 90
PI-91 ATCATATGTAGC CTCTACAGTCAC H07 91  PI-91 ATCATATGTAGC CTCTACAGTCAC H07 91
PI-92 TAGCATCGATAT AGATAGCACATC H08 92  PI-92 TAGCATCGATAT AGATAGCACATC H08 92
PI-93 TGATCGACGCTC CTAGATATCGTC H09 93  PI-93 TGATCGACGCTC CTAGATATCGTC H09 93
PI-94 TGCAGCTCATAG TACAGACTGCAC H10 94  PI-94 TGCAGCTCATAG TACAGACTGCAC H10 94
PI-95 CGACGTAGAGTC CAGTAGCACTAC Hll 95  PI-95 CGACGTAGAGTC CAGTAGCACTAC Hll 95
PI-96 CACTGTATAGCT CGACTAGTACTA H12 阴性对照 将步骤 1中以 KingFisher 自动提取仪提取的 DNA为模板， 以 5， 端带有标签的 HLA-C各外显子引物单管 PCR扩增， PCR程序如下： C2: 96 "C 2分钟； 95°C 30s 62°C 30秒 72°C 20秒 （35个循 环）； 15 °C oo ;  PI-96 CACTGTATAGCT CGACTAGTACTA H12 Negative Control The DNA extracted by the KingFisher Automatic Extractor in step 1 was used as a template, and the HLA-C exon primers with 5, end-end primers were PCR-amplified. The PCR procedure was as follows: C2 : 96 "C 2 minutes; 95 ° C 30s 62 ° C 30 seconds 72 ° C 20 seconds (35 cycles); 15 °C oo;
C3: 96 °C 2分钟； 95°C 30秒 56°C 30秒 72°C 20秒 （35个 循环）； 15°C∞; C4: 96 °C 2分钟； 95°C 30秒 60°C 30秒 72°C 20秒 （35个 循环）； 15°C∞。 C3: 96 ° C for 2 minutes; 95 ° C for 30 seconds 56 ° C for 30 seconds 72 ° C for 20 seconds (35 cycles); 15 ° C ∞; C4: 96 ° C for 2 minutes; 95 ° C for 30 seconds 60 ° C for 30 seconds 72 ° C for 20 seconds (35 cycles); 15 ° C ∞.
HLA-C的 PCR反应体系如下：  The PCR reaction system of HLA-C is as follows:
Figure imgf000011_0001
Figure imgf000011_0001
其中 PInrC-F2/3/4表示引物 5，末端带有第 n号正向引物标签序列 (表 2 ) 的 HLA-C的 F引物， PInrC-R2/3/4表示引物 5，末端带有第 n 号反向引物标签序列的 HLA-C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。 Wherein, PI nr CF 2/3/4 represents primer 5, HLA-C F primer with end number n forward primer tag sequence (Table 2), PI nr CR 2/3/4 represents primer 5, and end band The R primer of HLA-C having the nth reverse primer tag sequence (where n ≤ 96), and so on. And each sample corresponds to a specific set of PCR primers.
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显示了前 20个样本 HLA-C相应外显子 PCR产物电泳结果， DNA分子标记为 DL 2000 ( Takara公司） ， 胶图上有一系列片段大小为 400 bp-500 bp 单一条带， 表明这部分样本的 HLA-C各外显子 （C2、 C3、 C4 ) PCR 扩增成功。 其它样品的结果与此类似。  The PCR reaction was run on a Bio-Rad PTC-200 PCR machine. After the PCR was completed, 2 l of the PCR product was detected by 1.5% agarose gel electrophoresis. Figure 1 shows the results of electrophoresis of the corresponding exon PCR products of the first 20 samples of HLA-C. The DNA molecule is labeled DL 2000 ( Takara). The gel image has a series of fragments ranging from 400 bp to 500 bp. Some samples of HLA-C exons (C2, C3, C4) were successfully amplified by PCR. The results for the other samples are similar.
3. PCR产物混合和纯化  3. PCR product mixing and purification
从 96孔板 HLA-P-C2剩余的 PCR产物中 （阴性对照除外）各取 From the remaining PCR products of the 96-well plate HLA-P-C2 (except for the negative control)
20 μΐ混合在一个 3 ml的 ΕΡ管中， 标记为 HLA-C2-Mix, 对其它 2个 96孔板进行同样的操作， 分别标记为 HLA-C3-Mix和 HLA-C4-Mix, 震荡混匀，从 HLA-C2-Mix、 HLA-C3-Mix和 HLA-C4-Mix中各取 200 μΐ混合在一个 1.5 ml的 ΕΡ管中， 标记为 HLA-Mix， 从 HLA-Mix中 取 500 μΐ DNA混合物经 Qiagen DNA Purification kit ( QIAGE 公司） 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-Mix DNA浓 度为 50 ng l。 Mix 20 μΐ in a 3 ml tube, labeled HLA-C2-Mix, and perform the same operation on the other 2 96-well plates, labeled HLA-C3-Mix and HLA-C4-Mix, shake and mix 200 μL from HLA-C2-Mix, HLA-C3-Mix and HLA-C4-Mix in a 1.5 ml tube, labeled HLA-Mix, 500 μΐ DNA mixture from HLA-Mix Purified by a Qiagen DNA Purification kit (QIAGE) (specific purification steps are detailed in the instructions), purified 200 μl ϋΝΑ, and the HLA-Mix DNA concentration was determined to be 50 ng 1 by Nanodrop 8000 (Thermo Fisher Scientific).
4. Illumina GA PCR-Free测序文库的构建 4.1 PCR产物的打断 4. Construction of Illumina GA PCR-Free Sequencing Library 4.1 Interruption of PCR products
从纯化后的 HLA-Mix中取总量 5 μ8的 DNA 用 Covaris Take a total of 5 μ 8 of DNA from purified HLA-Mix with Covaris
microTube with AFA fiber and Snap - Cap在 Covaris S2(Covaris公司 ) 上打断。 打断条件如下： microTube with AFA fiber and Snap - Cap was interrupted on Covaris S2 (Covaris). The breaking conditions are as follows:
频率扫描 ( frequency sweeping )  Frequency sweeping
Figure imgf000012_0001
Figure imgf000012_0001
4.2 打断后的 PCR产物纯化  4.2 Purification of PCR products after interruption
将 HLA-Mix的所有打断产物用 QIAquick PCR Purification Kit 回收纯化， 分别溶于 37.5 μΐ的 EB ( QIAGEN Elution Buffer ) 中。  All interrupted products of HLA-Mix were recovered and purified by QIAquick PCR Purification Kit and dissolved in 37.5 μM EB (QIAGEN Elution Buffer).
4.3 末端修复反应  4.3 End repair response
对纯化的产物进行 DNA末端修复反应， 体系如下 （试剂均购自 DNA end-repair reaction of the purified product, the system is as follows (reagents are purchased from
Enzymatics公司 ) ： Enzymatics) :
Figure imgf000012_0002
Figure imgf000012_0002
反应条件为： 在 20 °C下， 在 Thermomixer(Eppendorf公司）中温 浴 30分钟。  The reaction conditions were as follows: at 20 ° C, in a Thermomixer (Eppendorf) bath for 30 minutes.
反应产物经 QIAqukk PCR Purification Kit回收纯化， 溶于 32 μΐ 的 EB ( QIAGEN Elution Buffer ) 中。  The reaction product was purified by QIAqukk PCR Purification Kit and dissolved in 32 μM EB (QIAGEN Elution Buffer).
4.4 3，末端加 A反应  4.4 3, end plus A reaction
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 ) ：  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):
Figure imgf000012_0003
Klenow (3'-5'exo-) 3 μΐ 总体积 50 μΐ 反应条件为： 在 37°C下， 在 Thermomixer中温浴 30分钟。
Figure imgf000012_0003
Klenow (3'-5'exo-) 3 μΐ Total volume 50 μΐ Reaction conditions: Warm bath in a Thermomixer for 30 minutes at 37 °C.
反应产物经 MiniElute PCR Purification Kit ( QIAGE 公司） 回 收純化， 溶于 38 μΐ的 ΕΒ溶液（ QIAGEN Elution Buffer ) 中。  The reaction product was purified by MiniElute PCR Purification Kit (QIAGE) and dissolved in a 38 μM solution (QIAGEN Elution Buffer).
4.5 连接 Illumina GA PCR-Free文库接头 （adaptor )  4.5 Connection Illumina GA PCR-Free Library Connector (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） ：  The product after addition of A was ligated to the Illumina GA PCR-Free library linker, and the system was as follows (the reagents were purchased from Illumina):
Figure imgf000013_0001
Figure imgf000013_0001
反应条件为： 在 16°C下， 在 Thermomixer中温浴过夜。  The reaction conditions were: overnight at a temperature of 16 ° C in a Thermomixer.
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_0002
The reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 μl of deionized water. The DNA concentration was determined by real-time PCR (QPCR) as follows:
Figure imgf000013_0002
4.6 割胶回收  4.6 tapping recycling
取 30μ1 HLA-Mix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 400-750 bp长度范围的 DNA片段（图 2 )。 胶回收 产物经 QIAquick PGR Purification Kit ( QIAGEN公司） 回收纯化， 纯化后体积为 32 μΐ, 经荧光定量 PCR ( QPCR )检测到 DNA浓度结 果为 17.16 nM。  30 μl of HLA-Mix was recovered using 2% low melting point agarose gel. The electrophoresis conditions were 100V for 100 minutes. The DNA standard molecular weight reference is a 50 bp DNA Ladder from NEB. The gel was recovered to recover DNA fragments ranging from 400-750 bp in length (Fig. 2). The gel recovery product was recovered and purified by QIAquick PGR Purification Kit (QIAGEN). After purification, the volume was 32 μΐ, and the DNA concentration was 17.16 nM by real-time PCR (QPCR).
5. Illumina GA测序  5. Illumina GA sequencing
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 II x ) 。  According to the QPCR test results, 10 pmol of DNA was sequenced using the Illumina GA PE-100 program. The specific procedure is detailed in the Illumina GA operating instructions (Illumina GA II x ).
6. 结果分析  6. Analysis of results
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The sequencing result of Illumina GA is a series of DNA sequences, sequenced by search The positive and negative primer tag sequences and primer sequences in the results, and the corresponding samples of each primer tag are established.
HLA-C各外显子 PCR产物测序结果的数据库； 通过 BWA HLA-C database of each exon PCR product sequencing result; by 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 (Burrows-Wheeler Aligner) locates 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 builds each database Consistency 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
( Pair-End连锁） 关系可组装成 HLA-C 各外显子相应的序列。 图 3 的截图示例性说明了对 1号样品的 HLA-C位点的 2号外显子一致性序 列进行构建的过程。  The (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-C. The screenshot of Figure 3 exemplifies the process of constructing the exon 2 consensus sequence for the HLA-C site of sample 1.
将所测序的 HLA-C内含子的 DNA序列与 IMGT HLA专业数据 库中 HLA-C 相应各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-C基因型别。 所有 95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号样本的具体结果与样本 原来分型结果对比如下： （如表 3所示， 全部检测结果与原有检测结 果相同） 。  The DNA sequence of the sequenced HLA-C intron is aligned with the sequence database of the corresponding exons of HLA-C in the IMGT HLA professional database, and the HLA-C gene of the corresponding sample is matched by 100% of the sequence alignment results. Type. For all 95 samples, the obtained classification results were completely consistent with the original known classification results. The specific results of the samples No. 1-32 were compared with the original classification results of the samples as follows: (As shown in Table 3, all the test results are the same as the original The test results are the same).
表 3. 本次分型结果与样本原来分型结果对比:  Table 3. Comparison of the results of this classification with the original classification of the sample:
Figure imgf000014_0001
21 C*03:03 C*08:01 C*03:03 C*08:01 是
Figure imgf000014_0001
21 C*03:03 C*08:01 C*03:03 C*08:01 Yes
22 C*03:04 C*07:02 C*03:04 C*07:02 是  22 C*03:04 C*07:02 C*03:04 C*07:02 Yes
23 C*07:02 C*08:01 C*07:02 C*08:01 是  23 C*07:02 C*08:01 C*07:02 C*08:01 Yes
24 C*07:02 C*12:02 C*07:02 C*12:02 是  24 C*07:02 C*12:02 C*07:02 C*12:02 Yes
25 C*07:02 C*12:03 C*07:02 C*12:03 是  25 C*07:02 C*12:03 C*07:02 C*12:03 Yes
26 C*03:04 C*08:01 C*03:04 C*08:01 是  26 C*03:04 C*08:01 C*03:04 C*08:01 Yes
27 C*01:02 C*03:04 C*01:02 C*03:04 是  27 C*01:02 C*03:04 C*01:02 C*03:04 Yes
28 C*07:02 C*12:02 C*07:02 C*12:02 是  28 C*07:02 C*12:02 C*07:02 C*12:02 Yes
29 C*03:02 C*07:02 C*03:02 C*07:02 是  29 C*03:02 C*07:02 C*03:02 C*07:02 Yes
30 C*01:02 C*03:03 C*01:02 C*03:03 是  30 C*01:02 C*03:03 C*01:02 C*03:03 Yes
31 C*01:02 C*07:02 C*01:02 C*07:02 是  31 C*01:02 C*07:02 C*01:02 C*07:02 Yes
32 C*01:02 C*07:02 C*01:02 C*07:02 是 注： HLA-C型别中的 C*0303不排除 C*0320N的可能性， C*0401不 排除 C*0409N/0430的可能性， C*0702不排除 C*0750的可能性， C*0801 不排除 C*0822的可能性， C*1505不排除 C*1529的可能性， 因为上述等 位基因在 HLA-C 2、 3、 4号外显子的序列完全相同。  32 C*01:02 C*07:02 C*01:02 C*07:02 Yes Note: C*0303 in HLA-C type does not exclude the possibility of C*0320N, C*0401 does not exclude C* The possibility of 0409N/0430, C*0702 does not exclude the possibility of C*0750, C*0801 does not exclude the possibility of C*0822, C*1505 does not rule out the possibility of C*1529, because the above allele is in HLA The sequences of exons 2, 3, and 4 are identical.
实施例 2. 用 Sanger法测序进行 HLA-C基因分型  Example 2. SLAG sequencing for HLA-C genotyping
1. 样品 DNA提取  Sample DNA extraction
如实施例 1中所述相似， 以 KingFisher自动提取仪提取的 95例 样本中的 26例已知 HLA基因型别的 DNA。  Similar to the one described in Example 1, 26 of the 95 samples extracted from the KingFisher automatic extractor were known to have HLA genotype DNA.
2. PCR扩增  2. PCR amplification
以上述 KingFisher自动提取仪提取的 DNA为模板， 以  Using the DNA extracted by the above KingFisher automatic extractor as a template,
C-F2,C-R2,C-F3,C-R3,C-F4,C-R4共 3对 PCR引物分别单管 PCR扩增， 各对引物 PCR程序如下：  C-F2, C-R2, C-F3, C-R3, C-F4, and C-R4 were amplified by single-tube PCR, and the PCR procedures of each pair of primers were as follows:
C2: 96 "C 2分钟; 95 "C 30秒 62 °C 30秒 - 72 °C 20秒 （35个 循环）； 15 °C oo ;  C2: 96 "C 2 minutes; 95 "C 30 seconds 62 °C 30 seconds - 72 °C 20 seconds (35 cycles); 15 °C oo;
C3: 96 °C 2分钟; 95 "C 30秒 -^56 "C 30秒 72。C 20秒 (35个 循环）； 15°C oo ;  C3: 96 ° C for 2 minutes; 95 "C 30 seconds - ^ 56 "C 30 seconds 72. C 20 seconds (35 cycles); 15 °C oo;
C4: 96 °C 2分钟; 95 "C 30秒 60°C 30秒 72 °C 20秒 （35个 循环）； 15 °C∞。  C4: 96 °C for 2 minutes; 95"C 30 seconds 60 °C 30 seconds 72 °C 20 seconds (35 cycles); 15 °C ∞.
HLA-C的 PCR反应体系如下：  The PCR reaction system of HLA-C is as follows:
Figure imgf000015_0001
DNA (约 20 ng/μΐ ) 2.0 μΐ
Figure imgf000015_0001
DNA (approx. 20 ng/μΐ) 2.0 μΐ
ddH20 12.8 μΐ ddH 2 0 12.8 μΐ
总计 25.0 μΐ  25.0 μΐ in total
PCR产物经琼脂糖凝胶电泳检测后 （图 4 ) , 准备纯化。  The PCR product was detected by agarose gel electrophoresis (Fig. 4) and prepared for purification.
3. PCR产物纯化  3. PCR product purification
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 μ1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 Pa, 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。  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 μl of ultrapure water to the wells to be used, attach the remaining membrane to the remaining pores, let stand for 15 minutes at room temperature or connect to the pumping On the filter system, take -10 Pa for 5 minutes. Each time the purification plate is removed from the suction system, the liquid remaining in the bottom drain of the purification plate is blotted on the absorbent paper.
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帕，抽滤至纯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。  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 μM 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 observed under illumination, not complete. The liquid surface reflects the luster.
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。  Add 50 μM ultrapure water or sputum 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 wells to the new 96-well PCR plate. Corresponding to the hole.
4. 进行测序反应并纯化测序反应产物  4. Perform sequencing reactions and purify sequencing reaction products
以上述纯化后的 PCR产物为模板做测序反应，测序反应的条件是： 96 °C 2分钟； 96°C 10秒 -> 55。C 5秒 60°C 2分钟 （25个循 环）； 15 "C ∞。  The sequencing reaction was carried out by using the above purified PCR product as a template, and the conditions for the sequencing reaction were: 96 ° C for 2 minutes; 96 ° C for 10 seconds -> 55. C 5 seconds 60 ° C 2 minutes (25 cycles); 15 "C ∞.
测序反应的体系是：  The system of sequencing reactions is:
Figure imgf000016_0001
Figure imgf000016_0001
通过以下步骤纯化测序反应产物：取下测序反应板配平，在 3000 g 下离心 1分钟。 96孔板每 5 μΐ反应体系加 2 μΐ 0.125 mol/L EDTA-Na2 溶液， 33 μΐ 85%乙醇， 盖上硅胶垫， 充分振荡 3分钟， 在 4°C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 50 μΐ 70%乙醇，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 10 μΐ ( 384孔板每孔加 8 μΐ ) HI-DI曱酰胺， 盖封 口膜， 振荡 5秒后离心至 1000 rpm。 The sequencing reaction product was purified by the following procedure: The sequencing reaction plate was removed and centrifuged at 3000 g for 1 minute. In a 96-well plate, add 2 μΐ 0.125 mol/L EDTA-Na2 solution, 33 μΐ 85% ethanol per 5 μΐ reaction solution, cover with a silica gel pad, and shake well for 3 minutes at 4 °C. Centrifuge at 3000 g for 30 minutes. 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. A 96-well plate was added with 50 μΐ 70% ethanol per well, covered with a silica gel 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 to an ethanol free odor. Add 10 μM per well to a 96-well plate (8 μM per well in a 384-well plate) HI-DI amide, cap the membrane, shake for 5 seconds, and centrifuge to 1000 rpm.
5. 测序和结果分析  5. Sequencing and results analysis
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 ) 。  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).
表 4. 本次分型结果与样本原来分型结果对比：  Table 4. Comparison of the results of this classification with the original classification of the sample:
Figure imgf000017_0001
Figure imgf000017_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。 It will be apparent to those skilled in the art that without departing from the scope and spirit of the invention Various modifications and changes can be made to the invention without departing from the scope of the invention. Other embodiments of the invention will be apparent to those skilled in the < 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.
参考文献=  References =
[1]. http://www.ebi.ac.uk/imgt/hla/stats.html  [1]. http://www.ebi.ac.uk/imgt/hla/stats.html
[2]. Tiercy J M. Molecular basis of HLA polymorphism:  [2]. Tiercy J M. Molecular basis of HLA polymorphism:
implications in clinical transplantation. Transpl Immunol, 2002, 9:Implication in clinical transplantation. Transpl Immunol, 2002, 9:
173-180. 173-180.
[3] Wegner KM: Massive parallel MHC genotyping: titanium that shines. Molecular Ecology 2009, 18: 1818-1820.  [3] Wegner KM: Massive parallel MHC genotyping: titanium that shines. Molecular Ecology 2009, 18: 1818-1820.
[4] Ellegren H: Sequencing goes 454 and takes large-scale genomics into the wild. Molecular Ecology 2008， 17: 1629-1635.  [4] Ellegren H: Sequencing goes 454 and takes large-scale genomics into the wild. Molecular Ecology 2008, 17: 1629-1635.
[5] Shendure J， Ji H: Next-generation DNA sequencing. Nature Biotechnology 2008, 26: 1135-1145.  [5] Shendure J, Ji H: Next-generation DNA sequencing. Nature Biotechnology 2008, 26: 1135-1145.
[6] Nagler A, B rautbar C, Slavin S, et al. Bone marrow  [6] Nagler A, B rautbar C, Slavin S, et al. Bone marrow
transplantation using unrelated and family related donors: the mpact of HLA-C disparity. Bone Marrow Transplant, 1996, 18 (5): 891-897. Transplanting using unrelated and family related donors: the mpact of HLA-C disparity. Bone Marrow Transplant, 1996, 18 (5): 891-897.
[7]Ho VT, Kim HT， Liney D， et al. HLA-C mismatch is associated with inferior survival after unrelated donor non-myeloablative hematopoietic stem cell transplantation. Bone Marrow Transplant, 2006, 37 (9):845-850.  [7] Ho VT, Kim HT, Liney D, et al. HLA-C mismatch is associated with inferior survival after unrelated donor non-myeloablative hematopoietic stem cell transplantation. Bone Marrow Transplant, 2006, 37 (9): 845-850.

Claims

权 利 要 求 书 Claim
1. 具有 SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5和 SEQ ID NO: 6任一项所示序列的多核苷酸。 A polynucleotide having the sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
2. 一种 HLA-C基因 2、 3和 /或 4号外显子扩增方法， 其特征在于 使用选自 SEQ ID NO: 1和 SEQ ID NO: 2; SEQ ID NO: 3和 SEQ ID NO: 4; 以及 SEQ ID NO: 5和 SEQ ID NO: 6的引物对进行 PCR扩 增， 扩增 HLA-C基因 2、 3和 /或 4号外显子。  2. A method for amplifying exon 2, 3 and/or 4 of HLA-C gene, characterized in that it is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; and the primer pairs of SEQ ID NO: 5 and SEQ ID NO: 6 were subjected to PCR amplification to amplify HLA-C gene exons 2, 3 and/or 4.
3. 一种对样品中 HLA-C基因 2、 3和 /或 4号外显子进行测序的方 法， 其包括步骤：  3. A method of sequencing HLA-C gene 2, 3 and/or exon 4 in a sample, comprising the steps of:
1 ) 提供一个样品并提取该样品的 DNA;  1) providing a sample and extracting DNA of the sample;
2 ) 将选自 SEQ ID NO: 1和 SEQ ID NO: 2； SEQ ID NO: 3和 SEQ ID NO: 4; 以及 SEQ ID NO: 5和 SEQ ID NO: 6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；  2) a primer pair selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; and SEQ ID NO: 5 and SEQ ID NO: 6 DNA thereby obtaining a PCR product, preferably purifying the PCR product;
3 ) 对所述 PCR产物进行测序。  3) Sequencing the PCR product.
4. 权利要求 3的方法， 其中所述样品是血样。  4. The method of claim 3, wherein the sample is a blood sample.
5. 权利要求 4的方法， 其中所述血样来自哺乳动物或人。  5. The method of claim 4, wherein the blood sample is from a mammal or a human.
6. 一种 HLA-C基因分型方法， 所述方法包括：  6. An HLA-C genotyping method, the method comprising:
1)使用选自 SEQ ID NO: 1和 SEQ ID NO: 2; SEQ ID NO: 3和 SEQ ID NO: 4; 以及 SEQ ID NO: 5和 SEQ ID NO: 6的引物对进行 1) using a primer pair selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; and SEQ ID NO: 5 and SEQ ID NO:
PCR扩增， 扩增待测样本的 HLA-C基因 2、 3和 /或 4号外显子； PCR amplification, amplification of the HLA-C gene 2, 3 and / or exon 4 of the sample to be tested;
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。  2) Sequencing the amplified exons and comparing the sequencing results with the standard sequences in the database to determine the genotyping results.
7. 权利要求 3或 6的方法， 其中所述测序是通过 Sanger测序法， 或者是通过第二代测序法。  7. The method of claim 3 or 6, wherein the sequencing is by Sanger sequencing or by second generation sequencing.
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。  8. The method of claim 7, wherein said second generation sequencing method is Illumina Solexa or Roche 454.
9. 一种用于进行 HLA-C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; SEQ ID NO: 3和 SEQ ID NO: 4; 以及 /或者 SEQ ID NO: 5和 SEQ ID NO: 6的 PCR扩增引物对， 所述 试剂盒优选还包含用于 DNA扩增、 DNA純化和 /或 DNA测序的试剂。  9. A kit for performing HLA-C genotyping, the kit comprising SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; The PCR amplification primer pair of SEQ ID NO: 5 and SEQ ID NO: 6, preferably further comprising reagents for DNA amplification, DNA purification and/or DNA sequencing.
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-C基因分型的用途。  10. Use of the polynucleotide of claim 1, the method of claim 6 or the kit of claim 9 for HLA-C genotyping.
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