WO2012000150A1 - Hla-a,b基因分型用pcr引物及其使用方法 - Google Patents
Hla-a,b基因分型用pcr引物及其使用方法 Download PDFInfo
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- WO2012000150A1 WO2012000150A1 PCT/CN2010/001813 CN2010001813W WO2012000150A1 WO 2012000150 A1 WO2012000150 A1 WO 2012000150A1 CN 2010001813 W CN2010001813 W CN 2010001813W WO 2012000150 A1 WO2012000150 A1 WO 2012000150A1
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the invention relates to the field of nucleic acid sequencing technology, in particular to the field of PCR sequencing technology.
- the present invention provides PCR primers for HLA-A, B (second generation sequencing) genotyping.
- the methods of the invention involve a method of typing DNA sequences, particularly a high resolution typing method for HLA genes. Background technique
- HLA Human leukocyte antigen
- PCR-SSP sequence-specific primer polymerase chain reaction
- PCR-SSO polymerase chain reaction oligonucleotide probe hybridization
- PCR-SBT polymerase chaining
- HLA-SSP The principle of HLA-SSP is to design a set of allele-specific primers, and obtain HLA type-specific amplification products by PCR technology, and determine the HLA type by electrophoresis analysis.
- the principle of HLA-SSO is to design an HLA-type specific oligonucleotide sequence as a probe, label the PCR product, and hybridize with the PCR product (gene DNA to be detected).
- the HLA type is determined by detecting the fluorescent signal.
- the detection signals of HLA-SSP and HLA-SSO are analog signals, and the resolution can only reach the low level and can not detect the new allele.
- HLA-SBT is a region related to HLA gene (HLA-A/B genotyping is generally simultaneous amplification of 2, 3, 4 exons for typing, and PCR products are all longer than lkb) PCR amplification
- the amplified 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.
- HLA-SBT's complex experimental procedures, low throughput and high cost make it difficult to apply to large-scale HLA high-resolution typing projects.
- HLA-SBT based on the second generation sequencing method represented by Illumina GA (Illumina's Genome Analyzer sequencer) and Roche 454 (Roche) (hereinafter referred to as the new sequencing technology) is also a DNA amplified by PCR.
- the product directly measures the nucleic acid sequence to determine the HLA genotype high-resolution typing method.
- it also has single-molecule sequencing characteristics.
- a primer primer can be synthesized by adding a primer index to the 5th end of the PCR primer. A unique primer label can be introduced into each sample during the PCR process, so that the sample can be used in the second.
- the detection result of each sample can be retrieved through its unique primer label sequence;
- the method has the characteristics of low cost, large flux and multiple different gene loci that can simultaneously detect a large number of samples.
- the DNA length of the sequencing library that can be used for the new sequencing technology can not be too long (Illunina GA's maximum applicable length is 700bp), plus The new sequencing technology is generally short, and the current Illumina GA bidirectional read length can only reach 200bp.
- the PCR primers originally used for the HLA-SBT method are no longer applicable. Combined with the characteristics of the new HLA sequencing technology, the length of the PCR product should not exceed 700 bp. Summary of the invention
- the PCR primers originally used for the HLA-SBT method are no longer suitable for the HLA high-resolution typing method based on the new sequencing technology.
- the present invention devised a novel set of PCR primers with specificity and conservation of the exons 2, 3 and 4 of the HLA- ⁇ , ⁇ gene, respectively, and the PCR product length is not more than 700 bp, which is especially suitable for Illumina. GA (currently the maximum DNA length for Illumina GA is 700bp).
- the set of PCR primers provided by the present invention can be used for large-scale, high-throughput and low-cost HLA genotyping of subjects, particularly humans.
- the technical solution adopted by the present invention is to plant all the latest HLA-A/B gene sequences from the IMGT/HLA Internet site (http://www.ebi.ac.uk/imgt/hla/) and then save them to the local disk.
- As the HLA-A data set simultaneously download all the latest non-HLA-A HLA-I 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, 3, and 4, and the designed PCR primer sequences were compared with the human genome sequence for homology.
- the HLA-A/B 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, and ensure that the primers amplify the HLA-A/B gene. Specificity.
- the length of the PCR product is less than 700 bp, and the annealing temperature of the positive and negative primers is substantially the same.
- the HLA- ⁇ , ⁇ -site primers designed by the present invention that is, the PCR primers for 6 pairs of HLA-A/B for amplifying exons 2, 3, and 4, respectively, are shown in Table 1.
- the invention provides a set of PCR primers for HLA-A, B genotyping, characterized in that the PCR primers are shown in Table 1.
- Another aspect of the invention provides a method for sequencing of the PCR primers shown in Table 1, comprising:
- the PCR primer is used to amplify DNA derived from a blood sample to obtain a PCR product, and the PCR product is purified;
- the PCR product is sequenced by Sanger sequencing or second generation sequencing (eg HiSeq 2000, Illumina GA and Roche454)
- Another aspect of the present invention provides the use of the PCR primers shown in Table 1 for HLA genotyping, characterized in that the PCR primers described above are used for assembly and alignment analysis according to the sequencing results obtained by the above methods, and the sequencing results are obtained.
- the HLA genotyping results were obtained by comparison with standard sequences in the database.
- the present invention provides a kit for performing HLA genotyping, which comprises the PCR primer of the present invention.
- a method of HLA typing comprising:
- n samples n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample;
- each pair of label primers are used, and in the presence of a template from the sample, PCR amplification is carried out under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of label primers comprises primers
- the label's forward label primer and reverse label primer (both may be degenerate primers), wherein the forward label primer and the reverse label primer may contain the same or different primer labels; the primers in the label primer pair used for different samples Labels are different from each other;
- mixing mixing: mixing PCR amplification products of each sample to obtain a PCR product library;
- the recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, IHumina GA), to obtain a sequence of the interrupted DNA;
- a second-generation sequencing technique preferably a Pair-End technique (eg, IHumina GA)
- the binding library linker technology constructing a PCR-free sequencing library from the disrupted PCR product library means using m library linkages to 4 a library of m PCR products obtained by adding a linker, wherein each PCR product library uses a different library linker to construct m linker tag sequencing libraries; m linker tag sequencing libraries are equimolarly mixed to construct a mixture Linker tag sequencing library.
- the method of ligating the library linker means that the DNA ligase is directly ligated without a PCR program.
- each A pair of primer tags and PCR primer pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer tag and a reverse primer tag, respectively.
- the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3 PCR primers for exon 4 and exon 2 of HLA-DRB1, preferably the PCR primers are shown in Table 3.
- Figure 1 The electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1. From the electropherogram, the PCR product is a series of single bands with a fragment size of 300bp-500bp, wherein lane M is the molecular weight marker. (DL 2000, Takara), Lanes 1-7 are HLA-A/B/DRB1 exons (A2, A3, A4, B2, B3, B4, DRBl-2) PCR amplification products , Negative control (N) without amplified bands. The results for the other samples are similar.
- Figure 2 DNA electrophoresis after HLA-Mix is interrupted (before and after tapping), and the tapping area is 450-750 bp.
- Lane M is a molecular weight marker (NEB-50bp DNA Ladder)
- lane 1 is the electrophoresis of HLA-Mix before tapping
- lane 2 is the gel of HLA-Mix after tapping.
- Figure 3 Screenshot of the sample consensus sequence constructor, which illustrates the splicing of PCR products based on the overlap between primer tags and DNA fragments. The complete sequence.
- a query for HLA typing naming can be found at http://www.ebi.ac.uk/imgt/hla/align.html. From the result output column on the left, we see the results of all coding sequences of A*02:03:01 A*ll:01:01, where the sequence of exon 2 is identical to the original known result of template No. 1. detailed description
- the PCR primers of the present invention and the PCR primers of the HLA-DRB1 exon 2 are used to perform HLA-A/B/DRB1 PCR amplification on 95 blood samples of common HLA genotypes.
- the amplified product was sequenced by IHumina GA Pair-End 100, and the sequencing results were obtained by assembly and comparative analysis.
- DNA was extracted from 95 blood samples of known HLA-SBT typing (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "Zhonghua Marrow Bank") using a KingFisher Automatic Extractor (Thermo Corporation, USA).
- the main steps are as follows: Take out the deep hole plate and one shallow hole plate of 6 Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them. Place all the well plates with reagents as required. In the corresponding position, select the program "Bioeasy_200ul Blood DNA-KF.msz” and press “star” to execute the program for nucleic acid extraction. At the end of the program, the lOOul elution product in the plate Elution is collected.
- PCR tag primers can be made by synthesizing PCR primers with different primer tags at the 5' end, such that different PCR tag primers can be used for different samples, the PCR primers are for HLA-A/B 2, 3, 4 Exon exon and PCR primers for exon 2 of HLA-DRB1. Primer tags are then introduced at both ends of the PCR product by PCR to specifically label PCR products from different samples.
- each set of PCR tag primers consisting of a pair of bidirectional primer tags (Table 2) and exons 2, 3, 4 for amplifying HLA-A/B and A PCR primer consisting of exon 2 of HLA-DRB1 (Table 3), wherein each forward PCR primer has a forward primer label attached to a pair of primer tags at the 5' end, and a 5' end of the reverse PCR primer is ligated.
- a reverse primer label for a pair of primer tags 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 of Example 1 were sequentially numbered 1-95, and the PCR reaction was carried out in a 96-well plate for 7 plates, numbered HLA-P-A2, HLA-P-A3, HLA. -P-A4, HLA-P-B2, HLA-P-B3, HLA-P-B4 and HLA-P-DRB1-2 (A2/3/4, B2/B3/B4, DRB1-2 for amplification) Site), a negative control without template was set in the plate, and the primer used in the negative control was the same as the corresponding primer of template 1. At the same time as the experiment, record the sample number information corresponding to each pair of primer labels.
- D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 are forward primers for amplifying HLA-DRB1 exon 2
- D2-R is for amplifying HLA- Reverse primer for exon 2 of DRB1.
- the PCR reaction system of HLA-A/B is as follows
- the PCR reaction system of HLA-DRB1 is as follows:
- PI nr A/B/D2-F 1/2 5/6/7 represents primer 5, and the F-primer of HLA-A/B/DRB1 with the nth forward primer tag sequence (Table 1) at the end, PI Nr A/B/D2-R 2/3/4 denotes primer 5, the R primer of HLA-A/B/DRB1 with the nth reverse primer tag sequence at the end (here n ⁇ 95 ), and so on .
- each sample corresponds to a specific set of PCR primers ( PI nr A/B/D2-F 1/2/3/4/s/6/7 PI n A/B/D2-R 2/3/4 )
- FIG. 1 shows the results of electrophoresis of the corresponding exon PCR product of sample No. 1 HLA-A/B/DRB1.
- the DNA molecular marker is DL 2000 ( Takara), and a series of fragments with a fragment size of 300bp-500bp are shown on the gel map.
- the HLA-A/B/DRB1 exons of sample No. 1 (A2 A3 A4 B2 B3 B4 DRB1-2 ) were successfully amplified by PCR, and the negative control (N) had no amplified bands. The results of other samples are similar to this.
- Example 3 Example 3
- HLA-A3-Mix HLA-A4-Mix HLA-B2-Mix HLA-B3-Mix, HLA-B4-Mi and HLA-D2-Mix oscillating and mixing from HLA-A2-Mix HLA-A3- Mix HLA-A4-Mix HLA-B2-Mix HLA-B3-Mix HLA-B4-Mix and 200 ul of HLA-D2-Mix was mixed in a 3 ml EP tube, labeled as HLA-Mix, and 500 ul of DNA mixture from HLA-Mix was purified by Qiagen DNA Purification kit (QIAGEN). See the instructions), 200 ul of DNA obtained was purified, and the concentration of HLA-Mix DNA was determined to be 48 ng/ul by Nanodrop 8000 (Thermo Fisher Scientific).
- QIAGEN Qiagen DNA Purification kit
- DNA end-repairing reaction of HLA-Mix purified after interruption such as The following (reagents are all purchased from Enzymatics):
- T4 DNA Polymerase (T4 DNA Polymerase) 5 ⁇ L ⁇
- the reaction conditions were: Thermomixer (Eppendorf) 20 warm bath 30 miii.
- reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 ⁇ 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):
- the reaction conditions were: Thermomixer 37 warm bath for 30 min.
- the reaction product was recovered and purified by a MiniElute PCR Purification Kit (QIAGE) and dissolved in a 13 ⁇ M solution (QIAGEN Elution Buffer).
- PCR-Free library adaptor refers to a designed base that is designed to assist in the immobilization of DNA molecules on sequencing chips and to provide universal sequencing primers.
- the binding site of the PCR-Free library linker can be directly ligated to the DNA fragment in the sequencing library by DN A ligase.
- the introduction process of the linker is called PCR-Free library linker because there is no PCR involved.
- PCR-free oligonucleotide linker mix (30mM) (PCR-free Adapter ⁇
- T4 DNA Ligase (Rapid, L603-HC-L) 3 ⁇ L ⁇ Total volume 30 ⁇ L
- the reaction conditions were: Thermomixer 20" C bath for 15 min.
- the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the DNA concentration was determined by real-time PCR (QPCR) as follows:
- HLA-Mix 30 L of HLA-Mix was recovered with 2% low melting point agarose gel. Electrophoresis conditions are 100V, 100min.
- the DNA marker is a 50 bp DNA marker from NEB.
- the taper recovers a DNA fragment of 450-750 bp in length (Fig. 2).
- the recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). After purification, the volume was 32 ul.
- the DNA concentration was 10.16 nM by real-time PCR (QPCR).
- the sequencing result of Illumina GA is a series of DNA sequences. By searching the sequence of the positive and negative primers in the sequencing results and the primer sequences, the sequencing results of each of the primers corresponding to the primers HLA-A/B/DRB 1 are established. Database. The sequencing results of each exon are mapped to the reference sequence of the corresponding exon by BWA (Burrows-Wheeler Aligner) (reference sequence source: http://www.ebi.ac.uk/imgt/hla/) Build consistency across databases
- the (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-A/B/DRB1.
- the resulting DNA sequence is aligned with the sequence database of the corresponding exons of HLA-A/B/DRB 1 in the IMGT HLA professional database, and the sequence alignment results are obtained.
- the 100% match is the HLA-A/B/DRB1 genotype of the corresponding sample.
- a screenshot of the exon 2 consensus sequence constructor for the HLA-A site of sample No. 1 illustrated in Figure 3 can be seen.
- Tiercy J M Molecular basis of HLA polymorphism: implications in clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180.
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Description
HLA-A, B基因分型用 PCR引物及其使用方法 技术领域
本发明涉及核酸测序技术领域, 特别是 PCR测序技术领域。 特别地, 本发明提供了用于 HLA-A,B (第二代测序法)基因分型 的 PCR引物。 另一方面, 本发明的方法涉及 DNA序列的分型方 法, 特别是 HLA基因高分辨率分型方法。 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***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***, 与同 种异体器官移植的排斥反应密切相关。 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。
目前国际标准的 HLA基因分型技术包括 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 是一种通过对 HLA基因的相关区域( HLA-A/B 基因分型一般同时扩增 2, 3, 4外显子用于分型, 其 PCR产物长 度都在 lkb以上)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。 HLA-SBT 整个实验流程复杂、 通量低和实猃成本高等 缺点使其很难应用于大规模 HLA高分辨分型项目。
基于以 Illumina GA ( Illumina公司的 Genome Analyzer测 序仪)和 Roche 454 ( Roche公司)为代表的第二代测序法(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, 还具有单分子测序特点。 其结合 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, 可在 PCR过程中对每个样本引入独特的引物标签, 使样本在利用第二代 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; 使该方法具有成本低, 通量大和可同时检测大量样本的多个 不同基因位点的特点。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 (Illunina GA的最大适用长度为 700bp ) , 再加上新测序技术读长普遍偏短, 当前 Illumina GA双向读长只 能达到 200bp, 原用于 HLA-SBT方法的 PCR引物不再适用。 结 合 HLA新测序技术的特点, PCR产物的长度不宜超过 700bp。
发明内容
鉴于现有新测序技术对 DNA模板长度的要求和新测序技术 读长偏短的事实,原用于 HLA-SBT方法的 PCR引物不再适用以 新测序技术为基础的 HLA 高分辨分型方法。 本发明设计了一套 全新的分别单独扩增 HLA-Α,Β基因的 2, 3, 4号外显子的特异 性和保守性良好的 PCR引物, 且 PCR产物长度不大于 700bp, 特别适用于 Illumina GA (当前 Illumina GA适用的最大 DNA长 度为 700bp )。本发明所提供的一套 PCR引物可用于对受试者(特 别是人) 进行大规模、 高通量和低成本的 HLA基因分型。
本发明采用的技术方案是, 从 IMGT/HLA 因特网站点 ( http://www.ebi.ac.uk/imgt/hla/ ) 下栽所有最新 HLA-A/B基因 序列, 然后保存到本地磁盘中做为 HLA-A数据集; 同时下载所 有最新非 HLA-A的 HLA-I类基因序列做为比较数据集。 将两数 据集进行比较, 在 2, 3, 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- A/B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 HLA-A/B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-A/B 引物用于扩增少数具 有 HLA-A/B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, 3, 4号外显子的 6对 HLA-A/B的 PCR引物。
用筛选出的 6对 PCR引物为基础引物, 并以此设计 95套标 签引物分别扩增 95个具有 HLA-A/B常见血清型的 DNA模板(该 部分模板的 HLA型别包括所有常见 HLA-A/B的血清型)。 所有 PCR产物等量混合后用 Illumina GA Pair-End 100测序, 组装后 的测序结果通过分型与原分型结果比较来验证 PCR 引物的保守 性和特异性。
本发明设计的 HLA-Α,Β位点引物, 即分别用于扩增 2, 3, 4 号外显子的 6对 HLA-A/B的 PCR引物见表 1。
表 1 HLA-Α,Β位点 PCR引物
提供样品, 特别是是血样, 所述血样优选地来自哺乳动物, 特别是人;
扩增: 使用所述 PCR引物用于扩增血样来源的 DNA从而得 到 PCR产物, 并对 PCR产物进行纯化;
测序: 对所述 PCR产物进行测序, 测序方法可以是 Sanger 测序法,或者可以是第二代测序法(例如 HiSeq 2000, Illumina GA 和 Roche454 )
本发明另一方面提供了表 1所示的 PCR引物用于 HLA基因 分型的用途,其特征在于使用上述的 PCR引物,根据上述方法得 到的测序结果进行组装和比对分析, 并将测序结果与数据库中的 标准序列进行比较, 得到 HLA基因分型结果。
另一方面, 本发明还提供了一种用于进行 HLA基因分型的 试剂盒, 所述试剂盒中包括本发明的 PCR引物。
本发明另一方面中, 提供了一种 HLA分型的方法, 其包括:
1 )提供 n个样品, n为大于等于 1的整数, 所述样品优选地来 自哺乳动物, 更优选是人, 特别是人的血样;
2 )将待分析的 n个样品分成 m个小组, m为整数且 n > m > l ;
3 )扩增: 对于每一个样品, 使用一对标签引物, 在存在来自 该样品的模板时, 在适于扩增目的核酸的条件下进行 PCR扩增, 其中, 每一对标签引物由包含引物标签的正向标签引物和反向标 签引物 (均可以是简并引物)构成, 其中正向标签引物和反向标 签引物所包含的引物标签可以相同或者不同; 不同样品所用标签 引物对中的引物标签彼此不同;
4 ) 混合: 将各样品的 PCR扩增产物混合在一起, 获得 PCR 产物文库;
5 )打断: 将所得的 PCR产物文库进行不完全打断;
6 )建库: 结合文库接头标签技术, 将打断后的 PCR产物文库 构建 PCR-Free测序文库, 回收位于所用测序仪最大读长长度到所 用测序仪适用的最长 DNA长度范围之间的所有 DNA条带, 可以对 文库添加不同的文库接头 (adapter ) 以区分不同的 PCR-Free测 序文库;
7 ) 测序: 将回收的 DNA混合物利用二代测序技术, 优选的 是 Pair-End技术(例如 IHumina GA )进行测序, 获得打断后的 DNA的序列;
8 )拼接:基于各个文库不同的文库接头序列和每个样品独特 的引物标签将获得的测序结果与样品一一对应,利用比对程序(例 如 Blast,BWA程序) ^各个测序序列定位到 PCR产物的相应 DNA 参考序列上,通过序列重叠和连锁关系, 从打断后的 DNA的序列 拼接出完整的目的核酸。
在本发明的一个具体实施方式中, 在上文所述的方法中, 所 述结合文库接头标签技术, 将打断后的 PCR 产物文库构建 PCR-Free测序文库是指使用 m种文库接头给 4 ) 中得到的 m个 PCR产物文库加上接头, 其中每一个 PCR产物文库使用一种不 同的文库接头, 从而构建 m个接头标签测序文库; 将 m个接头 标签测序文库等摩尔混合在一起构建混合接头标签测序文库。 其 中连接文库接头的方法是指不通过 PCR程序直接采用 DNA连接 酶进行连接。
在本发明的一个具体实施方式中, 在上文所述的方法中, 每
一对引物标签与 PCR引物对组合成一对标签引物, 正反 PCR引 物的 5,端分别具有 (或者任选通过连接序列连接)正向引物标签 和反向引物标签。
在本发明的一个具体实施方式中, 在上文所述的方法中, 所 述 PCR引物是用于扩增 HLA的特定基因的 PCR引物, 优选是 用于扩增 HLA-A/B 2, 3, 4号外显子和 HLA-DRB1 2号外显子 的 PCR引物, 优选的所述 PCR引物如表 3所示。
本发明所提供的一套用于 HLA基因分型的 PCR引物, 其中
1 ) 6对 PCR引物扩增 HLA-A、 B两位点 2, 3, 4号外显子 的序列, PCR产物长度均在 700bp以内。
2 ) 引物保守性良好和特异性良好。 附图说明
图 1: 为 1号样本 HLA-A/B/DRB1相应外显子 PCR产物电泳 结果, 从电泳图上看, PCR 产物为一系列片段大小 300bp-500bp 的单一条带,其中泳道 M是分子量标记物( DL 2000,Takara公司), 泳道 1-7为 1号样本的 HLA-A/B/DRB1各外显子 ( A2、 A3、 A4、 B2、 B3、 B4、 DRBl-2 ) PCR扩增产物, 阴性对照 (N )无扩增条 带。 其它样品的结果与此类似。
图 2: 为 HLA-Mix打断后 DNA电泳情况 (割胶前后), 割胶 区域为 450-750bp区域。 其中泳道 M是分子量标记物(NEB-50bp DNA Ladder ) , 泳道 1是割胶前 HLA-Mix的电泳情况, 泳道 2 是割胶后 HLA-Mix的胶图。
图 3: 1号样本一致性(consensus )序列构建程序截图, 示例 说明了根据引物标签和 DNA片段之间的重叠关系拼接出 PCR产物
的 完整序 列 。 关 于 HLA 分型 命名 的 查询 可见于 http://www.ebi.ac.uk/imgt/hla/align.html。 从左侧结果输出栏里面 我们看到了 A*02:03:01 A*ll:01:01的所有编码序列的结果,其中 2号外显子的序列与 1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, 但是 本领域技术人员将会理解, 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。
在本发明的实施例中,采用本发明的 PCR引物和 HLA-DRB1 2号外显子的 PCR引物,对 95例已知 HLA常见基因型别的血样 进行 HLA-A/B/DRB1位点 PCR扩增, 扩增产物经 IHumina GA Pair-End 100测序, 测序结果经过组装和比对分析得到样品的分 型结果。 实施例 1
样本提取
使用 KingFisher 自动提取仪(美国 Thermo公司)从 95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 (以下称 "中华骨髓库" )) 中提取 DNA。 主要步驟如下: 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_200ul Blood DNA一 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 lOOul左右的洗脱产物即为提
取的 DNAo 实施例 2
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-A/B的 2, 3, 4号外显子以及 HLA-DRB1 2号外显子的 PCR引物。其后通过 PCR反应在 PCR 产物两端引入引物标签,从而特异地标记了来自不同样本的 PCR 产物。
以 95套 PCR标签引物来分别扩增 95份 DNA样本,每套 PCR 标签引物由一对双向引物标签(表 2 )和用于扩增 HLA-A/B的 2, 3, 4号外显子以及 HLA-DRB1 2号外显子的 PCR引物 (表 3 ) 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。
把实施例 1的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 7板,编号分别为 HLA-P- A2、 HLA-P-A3, HLA-P-A4、 HLA-P-B2、 HLA-P-B3, HLA-P-B4以 及 HLA-P-DRB1-2 ( A2/3/4, B2/B3/B4, DRB1-2 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 与模板 1的对应引物相同。 实验的同时, 记录下每对引物标签对 应的样本编号信息。
£18T00/010ZN3/X3d OSTOOO/ZIOZ OAV
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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 Hl l 95
表 3, 未添加引物标签前用于扩增 HLA-A/B/DRB1相应外显子 的 PCR引物
引物编号 引物序列 引物用途 产物长度
A-F2 CCTCTGYGGGGAGAAGCAA 扩 HLA-A基因 2
480bp
A-R2 ATCTCGGACCCGGAGACTG 号外显子
A-F3 CGGGGCCAGGTTCTCACAC 扩 HLA-A基因 3
410bp
A-R3 GGYGATATTCTAGTGTTGGTCCCAA 号外显子
A-F4 GTGTCCCATGACAGATGCAAAA 扩 HLA-A基因 4
430bp
A-R4 GGCCCTGACCCTGCTAAAGG 号外显子
B-F2 AGGAGCGAGGGGACCGCA 扩 HLA-B基因 2
400bp
B-R2 CGGGCCGGGGTCACTCAC 号外显子
B-F3 CGGGGCCAGGGTCTCACA 扩 HLA-B基因 3
370bp
B-R3 GAGGCCATCCCCGGCGAC 号外显子
B-F4 GCTGGTCACATGGGTGGTCCTA 扩 HLA-A基因 4
380bp
B-R4 CTCCTTACCCCATCTCAGGGTG 号外显子
D2-F1 CACGTTTCTTGGAGTACTCTA
D2-F2 GTTTCTTGTGGCAgCTTAAgTT
D2-F3 CCTGTGGCAGGGTAAGTATA
D2-F4 GTTTCTTGAAGCAGGATAAGTT 扩 HLA-DRB1基
300bp
D2-F5 GCACGTTTCTTGGAGGAGG 因 2号外显子
D2-F6 TTTCCTGTGGCAGCCTAAGA
D2-F7 GTTTCTTGGAGCAGGTTAAAC
D2-R CCTCACCTCGCCGCTGCAC
D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 为扩增 HLA-DRBl 2号外显子的正向引物, D2-R为扩增 HLA-DRBl 2号 外显子的反向引物。
HLA-A/B/DRB1 的 PCR程序如下:
96 X: 2min
95 X: 30s ->60X 30s ^72 X: 20s (32cycles) 15X oo
HLA-A/B的 PCR反应体系如下
HLA-DRBl的 PCR反应体系如下:
DNA (约 20 ng/ul ) 5.0ul
ddH20 4.8ul
Total 25.0ul
其中 PInrA/B/D2-F1/2 5/6/7表示引物 5,末端带有第 n号正向引 物标签序列(表 1 )的 HLA-A/B/DRB1的 F引物, PInrA/B/D2-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-A/B/DRB1 的 R引物(此处 n < 95 ) , 其它依次类推。 且每个样本对应特定的 一套 PCR引物 ( PInrA/B/D2-F1/2/3/4/s/6/7 PIn A/B/D2-R2/3/4 )
PCR反应在 Bio-Rad公司的 PTC-200 PCR仪上运行。 PCR完 成后, 取 2ul PCR产物经 1%的琼脂糖凝胶电泳检测。 图 1显示 了 1号样本 HLA-A/B/DRB1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 300bp-500bp单一条带, 表明 1号样本的 HLA-A/B/DRB1各外显 子 (A2 A3 A4 B2 B3 B4 DRB1-2 ) PCR扩增成功, 阴性 对照 (N )无扩增条带。 其它样品的结果与此类似 实施例 3
PCR产物混合和纯化
从 96孔板 HLA-P-A2剩余的 PCR产物中 (阴性对照除外) 各取 20ul混合在一个 3ml的 EP管中, 标记为 HLA-A2-Mix, 对 其它 6个 96孔板进行同样的操作, 分别标记为 HLA-A3-Mix HLA-A4-Mix HLA-B2-Mix HLA-B3-Mix , HLA-B4-Mi 和 HLA-D2-Mix , 震荡混匀 从 HLA-A2-Mix HLA-A3-Mix HLA-A4-Mix HLA-B2-Mix HLA-B3-Mix HLA-B4-Mix 和
HLA-D2-Mix中各取 200ul混合在一个 3ml的 EP管中, 标记为 HLA-Mix, 从 HLA-Mix中取 500ul DNA混合物经 Qiagen DNA Purification kit ( QIAGEN公司) 过柱纯化 (具体纯化步骤详见 说明书) , 纯化所得的 200ul DNA, 经 Nanodrop 8000(Thermo Fisher Scientific公司)测定 HLA-Mix DNA浓度为 48ng/ul。 实施例 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-Mix 中取总量 5ug 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 )
2. 打断后纯化
将 HLA-Mix的所有打断产物用 QIAquick PCR Purification Kit回收纯化, 分别溶于 37.5ul的 EB ( QIAGEN Elution Buffer ) 中;
3. 末端修复反应
对打断后纯化的 HLA-Mix进行 DNA末端修复反应, 体系如
下 (试剂均购自 Enzymatics公司) :
DNA 37.5μί
H20 37.5μί 多核苷酸激酶緩冲液( 10x Polynucleotide Kinase Bufferi B904 ) ) 10 dNTP混合物 (每种 lOmM ) ( Solution Set ( lOmM each ) ) 4
T4 DNA聚合酶( T4 DNA Polymerase ) 5μL·
Klenow片段 ( Kleno Fragment )
T 多聚核苷酸激酶( T4 Polynucleotide Kinase ) 5μL 总体积 ( Total volume ) 100
反应条件为: Thermomixer(Eppendorf 公司) 20 温浴 30 miii。
反应产物经 QIAquick PCR Purification Kit回收纯化, 溶于 34 μΐ的 EB ( 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 ) :
上一步所得 DNA 32
10x 蓝色緩沖液 ( 10x blue buffer ) 5 μL· dATP(lmM, GE公司) 10 μΐ
Klenow (3'-5' exo-) 3 总体积 ( Total volume ) 50
反应条件为: Thermomixer 37 温浴 30 min。
反应产物经 MiniElute PCR Purification Kit( QIAGE 公司) 回收纯化, 溶于 13 μΐ的 ΕΒ溶液( QIAGEN Elution Buffer ) 中。
5. 连接 Illumina GA PCR-Free文库接头 ( adaptor ) 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 DN A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。
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) :
上一步所得 DNA Ι ΐμί
2\ 快速连接緩冲液 ( 2x Rapid ligation buffer )
PCR-free寡核苷酸接头混合物 (30mM) ( PCR-free Adapter Ιμί
oligo mix )
T4 DNA连接酶( T4 DNA Ligase ) (Rapid , L603-HC-L) 3μL· 总体积 ( Total volume ) 30 μL
反应条件为: Thermomixer 20"C温浴 15 min。
6. 割胶回收
取 30 L HLA-Mix用 2%低熔点琼脂糖胶进行回收。 电泳条件为
100V, 100min。 DNA marker为 NEB公司的 50bp DNA marker. 割胶回收 450-750bp长度范围的 DNA片段(附图 2 ) 。 胶回收产 物经 QIAquick PCR Purification Kit ( QIAGEN公司)回收纯化, 纯化后体积为 32ul, 经荧光定量 PCR ( QPCR )检测到 DNA浓 度结果为 10.16nM。 实施例 5
Illumina GA测序
根据 QPCR检测结果, 取 lOpmol 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 ) 。 实施例 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- A/B/DRB 1 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/ ) 同时, 构建各个数据库的一致性
( consensus )序列, 再对数据库中 DNA序列进行筛选和测序错误 校正。 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-A/B/DRB1 各外显子相应的 序列 。 所得 DNA序列利用 与 IMGT HLA专业数据库中 HLA-A/B/DRB 1 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-A/B/DRB1基因型别。可参考图 3示例说明的 1号样品的 HLA-A位点的 2号外显子一致性序列构建 程序的截图。
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号样本的具体结果如下:
样本编号 原 -A/B/DRB1型别
1 A*02:03 A*ll:01 B' k38:02 B*48:01 DRB1 *14:54 DRB1*15:01
2 A*01:01 A*30:01 B' k08:01 B' fc13:02 DRB1 "03:01 DRB1*07:01
3 A*01:01 A*02:01 B' "15:11 B' fe47:01 DRB1' *13:02 DRB1*15:01
4 A*24:08 A*26:01 B*40:01 B' fe51:01 DRB1 fc04:04 DRB1*09:01
5 A*01 :01 A*24:02 B' k 54:01 B' "=55:02 DRB1' *04:0S DRB1*09:01
6 A*01 :01 A*03:02 Bj 45:11 B' 7:01 DRBl' "10:01 DRB1*14:54
7 A*ll:01 A*30:01 B' k13:02 B' k15:18 DRB1' fc04:04 DRB1*07:01
8 A*01 :01 A*02:01 B*35:03 B' k81 :01 DRBl' kll:01 DRB1*15:01
9 A*02:06 A*31:01 Bj ^27:07 Bj 40:02 DRBl' "=03:01 DRB1*13:02
10 A*01 :01 A*66:01 Bj 37:01 49:01 DRBl' "=10:01 DRB1*13:02
11 A*01:01 A*03:01 35:01 K52:01 DRBl' ^OI JOI DRB1*15:02
12 A*ll:01 A*ll:01 BJ k15:01 BJ 45:05 DRBl' "04:06 DRB1H5:01
13 A*01:01 A*ll:02 BJ k07:02 45:02 DRBl' ^09:01 DRB1*15:01
14 A*01:01 A*02:01 Bj k52:01 ^67:01 DRBl' "=15:02 DRB1*16:02
15 A*01:01 A*02:05 Bj 5:17 k50:01 DRBl' k07:01 DRB1*15:01
16 A*01:01 A*ll:01 BJ k37:01 BJ k 40:02 DRBl' •=10:01 DRB1 *12:02
17 A*24:07 A*32:01 BJ 35:05 BJ k 40:01 DRBl' "03:01 DRB1*04:05
18 A*ll:01 A*24:02 43:01 Bj k35:01 DRBl' 46:02 DRB1*16:02
19 A*ll:01 A*ll:01 40:02 k55:12 DRBl' "04:05 DRB1*15:01
20 A*02:ll A*24:02 B*40:01 Bj fc40:06 DRBl' 41:01 DRB1*15:01
21 A*01:01 A*02:06 fc51:01 B" k57:01 DRBl^ k07:01 DRB1*12:01
22 A*01:01 A*29:01 B*07:05 45:01 DRBl ' ^04:05 DRB1*07:01
23 A*01:01 A*02:07 '37:01 46:01 DRB1J ^04:03 DRB1*10:01
24 A*24:85 A*30:01 B" 13:02 BV 5:02 DRB1 J K07:01 DRB1*15:01
25 A*ll:01 A*31:01 ^07:06 1:01 ORBV 42:02 DRB1*14:05
26 A*01:01 A*ll:01 '46:01 B" '57:01 ORBV k07:01 DRB1*08:03
27 A*01:01 A*02:01 B*15:18 B" '37:01 ORBV ^4:01 DRB1*15:01
28 A*01:01 A*24:02 B" 37:01 B" '46:01 ORBV k09:01 DRB1*10:01
29 A*26:01 A*66:01 B*40:40 B*41:02 ORBV k12:01 DRB1*15:01
30 A*02:01 A*29:02 B" 13:02 45:01 ORBV k03:01 DRB1 *12:02
31 A*01:01 A*ll:03 15:01 57:01 ORBV ^07:01 DRB1*15:01
32 A*ll:01 A*26:01 B*35:03 B *38:01 DRB1! "11:03 样本编号 测得的 HLA-A/B/DRBl型别
1 A*02:03 A*ll:01 B' "3S:02 B fc48:01 DRB1' fc14:54 DRB1*15:01
2 A*01:01 A*30:01 B*08:01 B*13:02 DRB1' ¾03:01 DRB1*07:01
3 A*01:01 A*02:01 B' k15:ll B*47:01 DRBl^ ^ 13:02 DRB1*15:01
4 A*24:08 A*26:01 B' k 40:01 B' fc51:01 DRB1' ^04:04 DRB1*09:01
5 A*01:01 A*24:02 B' "=54:01 B *55:02 DRB1' ¾ 04:05 DRB1 *09:01
6 A*01:01 A*03:02 B' "15:11 B' k37:01 DRB1J fe10:01 DRB1*14:54
7 A*ll:01 A*30:01 B' "13:02 B' "15:18 DRBl j ^04:04 DRB1*07:01
8 A*01:01 A*02:01 B' 5:03 B' k81:01 DRBlj 1:01 DRB1*15:01
9 A*02:06 A*31:01 B*27:07 B' 0:02 DRBV ^03:01 DRB1 *13:02
10 A*01 :01 A*66:01 B*37:01 B*49:01 DRBV k10:01 DRB1*13:02
11 A*01:01 A*03:01 "35:01 B' "52:01 ORBV ^1:01 DRB1*15:02
12 A*ll:01 A*ll:01 Bj 5:01 B' 45:05 DRBV ^04:06 DRB1*15:01
13 A*01:01 A*ll:02 B' ^7:02 B' 45:02 DRBV fc09:01 DRB1*15:01
14 A*01:01 A*02:01 k52:01 B' ^67:01 DRBV 45:02 DRB1*16:02
15 A*01:01 A*02:05 ' 45: 17 ^50:01 DRBV k07:01 DRB1*15:01
16 A*01:01 A*ll :01 k37:01 B' k 40:02 DRBV 0:01 DRB1*12:02
17 A*24:07 A*32:01 k35:05 B' k 40:01 ORBV '03:01 DRB1*04:05
18 A*l l:01 A*24:02 BJ k13:01 fe35:01 DRBV = 16:02 DRB1*16:02
19 A*ll:01 A*ll:01 Bj fc 40:02 B' fe55:12 DRBV ^04:05 DRB1*15:01
20 A*02:l l A*24:02 Bv k40:01 40:06 ORBV 41:01 DRB1*15:01
21 A*01:01 A*02:06 BJ k51:01 BJ k57:01 DRB1" (07:01 DRB1*12:01
22 A*01:01 A*29:01 k07:05 Bj 45:01 DRB1" '04:05 DRB1 *07:01
23 A*01 :01 A*02:07 k37:01 fe46:01 DRB1" =04:03 DRB1*10:01
24 A*24:85 A*30:01 13:02 BJ 5:02 DRBV '07:01 DRB1*15:01
25 A*ll:01 A*31:01 B*07:06 k51:01 DRBV 12:02 DRB1*14:05
26 A*01:01 A*ll:01 B" '46:01 k57:01 ORBV '07:01 DRB1*08:03
27 A*01:01 A*02:01 B" '15:18 37:01 DRB1*04:01 DRB1*15:01
28 A*01:01 A*24:02 B' '37:01 B' k 46:01 DRB 09:01 DRB1 *10:01
29 A*26:01 A*66:01 40:40 =41:02 DRB1* 12:01 DRB1*15:01
30 A*02:01 A*29:02 B* '13:02 B" '45:01 DRBl* 03:01 DRB1*12:02
31 A*01:01 A* 11 :03 B* 15:01 7:01 DRB1* 07:01 DRB1*15:01
32 A*ll:01 A*26:01 B*35:03 '38:01 DRB1 " 11:03 DRB1*14:04 注 : HLA-DRBl 型 别 中 的 DRB1* 1201 不 排 除
DRB1*1206/1210/1217的可能性, DRB1*1454不排除 DRB1*1401
的可能性, 因为上述等位基因在 HLA-DRB1 2号外显子的序列完 全相同。 尽管本发明的具体实施方式已经得到详细的描述, 本领域技 术人员将会理解。 根据已经公开的所有教导, 可以对那些细节进 行各种修改和替换, 这些改变均在本发明的保护范围之内。 本发 明的全部范围由所附权利要求及其任何等同物给出。 参考文献
[1]. http://www.ebi.ac.ukyimgt/hla/stats.html
[2]. Tiercy J M. Molecular basis of HLA polymorphism: implications in clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180.
[3]. C.Antoine, S.Miiller, A.Cant, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience. 1968-99.
[J], The Lancet, 2003,9357:553-560.
[4]. H. A. Erlich, G. Opelz, J. Hansen, et al. HLA DNA Typing and Transplantation.
[J].Immunity, 2001,14:347-356.
[5]. Lillo R, Balas A, Vicario JL, et al. Two new HLA class allele, DPBl*02014,by sequence-based typing. [J]. Tissue Antigens, 2002, 59: 47-48.
[6]. A. Dormoy, N. Froelich . Leisenbach, et al. Mono-allelic amplification of exons 2-4 using allele group-specific primers for
sequence-based typing (SBT) of the HLA-A, -B and -C genes: Preparation and validation of ready-to-use pre-SBT mini-kits. [J]. Tissue Antigens, 2003, 62: 201-216.
[7], Elaine R. Mardis. The impact of next-generation sequencing technology on genetics. [J]. Trends in Genetics.2008,24:133-141.
[8]. Christian Hoffmannl, Nana Minkahl, Jeremy Leipzig. DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations. [J]. Nucleic Acids Research, 2007,1-8
[9]. Sayer D, Whidborne R, Brestovac B. HLA-DRBl DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors. [J]. Tissue Antigens. 2001, 57(l):46-54.
Claims
1. 一套用于 HLA-A,B基因分型的 PCR引物, 其特征在于所 述 PCR引物如表 1所示,优选地所述分析是基于测序法, 包括第 二代测序法, 和 Sanger法。
2. 权利要求 1所述的 PCR引物的用于测序的方法, 其包括: 提供样品, 特别是血样, 所述血样优选地来自哺乳动物, 特 别是人;
扩增: 使用所述 PCR引物用于扩增血样来源的 DNA从而得 到 PCR产物, 并对 PCR产物进行纯化;
测序: 对所述 PCR产物进行测序, 测序方法可以是 Sanger 测序法,或者可以是第二代测序法(例如 HiSeq 2000、 Illumina GA 和 Roche454 ) 。
3. 权利要求 1所述的 PCR引物用于 HLA基因分型的用途, 其特征在于使用权利要求 1 所述的 PCR 引物, 根据权利要求 2 的方法得到的结果进行组装和比对分析, 并将测序结果与数据库 中的标准序列进行比较, 得到 HLA基因分型结果。
4. 一种用于进行 HLA基因分型的试剂盒, 所述试剂盒中包 括权利要求 1所述的 PCR引物。
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CN101921841B (zh) * | 2010-06-30 | 2014-03-12 | 深圳华大基因科技有限公司 | 基于Illumina GA测序技术的HLA基因高分辨率分型方法 |
CA2803940C (en) | 2010-06-30 | 2019-07-02 | Bgi Shenzhen Co., Limited | Application of a pcr sequencing method, based on dna barcoding technique and dna incomplete shearing strategy, in hla genotyping |
CN103261438B (zh) * | 2010-12-24 | 2015-09-16 | 深圳华大基因医学有限公司 | Hla-c基因分型的方法及其相关引物 |
CN107937487B (zh) * | 2017-12-29 | 2019-06-21 | 北京诺诗康瀛基因技术股份有限公司 | 一种用于hla-a基因pcr扩增、基因分型的方法、引物组及试剂盒 |
CN108441547B (zh) * | 2018-04-13 | 2020-02-07 | 北京诺诗康瀛基因技术股份有限公司 | 一种hla基因扩增、基因分型的引物组、试剂盒和方法 |
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CN112921078B (zh) * | 2021-02-08 | 2022-07-08 | 杭州虎贲生物医疗有限公司 | 一种测定待测者的hla-i分型的方法和一种锚序列组 |
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