CN103261438A - Method for hla- genotyping and related primers thereof - Google Patents
Method for hla- genotyping and related primers thereof Download PDFInfo
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- 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
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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
The method and its relevant primer technical field of HLA-C Genotypings specifically, the present invention relates to the method for HLA-C Genotypings the present invention relates to biology field, and the specific primer used in methods described.Background technology HLA (human leucocyte antigen, HLA) is the important component of human immune system, is also current known human body most complicated genetic polymorphism sexual system.The characteristics of according in terms of its structure, Tissue distribution and function, HLA molecules can be divided into the quasi-molecule of HLA- I, HLA- II and HLA-m etc. three, wherein HLA- I quasi-molecules and HLA- II quasi-molecule functions is mainly related to immunological rejection, and HLA-m quasi-molecules function is mainly related to the synthesis of Ia part complement system and inflammation-related factor etc.；Clinical transplantation research is found, the long-term surviving rate of graft and the being proportionate property of matching degree of donor and HLA- I and HLA- the II molecules of receptor both sides, when the matching degree for the HLA molecules by both sides is higher, the long-term surviving rate of graft is higher.
Wherein HLA-C sites belong to classical HLA- I genoids, between HLA-A, B site, encode HLA-C molecules, and its mrna length about 3Kb is made up of 7 intrones and 8 extrons.In recent years, continuing to develop with HLA genotyping techniques, people gradually have found HLA-C as HLA-A, B molecule, with high polymorphism, and confirm importance of the HLA-C molecules in clinical transplantation.
The HLA typing methods of current international standard include PCR-SSP (sequence specific primers PCR), PCR-SSO (PCR oligonucleotide probe hybridization) and PCR-SBT (PCR product sequencing based types）.
HLA-SSP principle is to design a whole set of allele group-specific primers, obtains the special amplified production of HLA types by round pcr, HLA types are determined by electrophoretic analysis.HLA-SSO principle is to design the special oligonucleotide sequence of HLA types as probe, PCR primer is marked, with PCR primer（Gene DNA to be detected) hybridize with probe.By detecting that fluorescence signal judges HLA types.HLA-SSP and HLA-SSO detection signal is analog signal, and low-level and new allele can not be all detected during resolution ratio can only typically be reached.
Based on Sanger methods be sequenced HLA-SBT be it is a kind of by being expanded to PCR after
DNA product directly carries out Sanger method sequencings（Capillary Ventral medulla）, nucleotide sequence is determined, so that judge the other high-resolution classifying method of HLA genotype, the characteristics of it has directly perceived, high-resolution and can detect new allele.But the shortcomings of based on the whole experiment flow complexity of HLA-SBT, low flux and the high experimental cost that Sanger methods are sequenced, makes it difficult to be applied to extensive HLA high-resolution parting project.
Based on second generation sequencing technologies (hereinafter referred to as new sequencing technologies of the Illumina Solexa and Roche 454 for representative）HLA-SBT be also it is a kind of by being expanded to PCR after DNA product directly determine nucleotide sequence, so as to judge the other high-resolution classifying method of HLA genotype, its in addition to original directly perceived, high-resolution and the characteristics of neomorph can be detected, also with single-molecule sequencing, simple experiment flow, high flux and low cost the characteristics of.But with first generation sequencing technologies（Sequencing technologies based on Sanger methods sequencing principle）Compare, the DNA length that can be used for new sequencing technologies sequencing library preparation can not be oversize（Current Illumina Solexa maximum prove-in length is 700bp), and new sequencing technologies read long universal partially short, maximum is grown in the current two-way readings of Illumia GA can reach 300bp.
In view of the characteristics of new sequencing technologies, the length of PCR primer is no more than 700bp, and former base is no longer applicable in the HLA-SBT of Sanger method sequence measurements PCR primer.Therefore, it is necessary to design the primer that a set of conservative meets new sequencing technologies requirement with specific good and PCR primer length.
Bibliography
[1] Marsh, S.G.E., Parham, P. & Barber, L.D. The HLA Facts
Book 3-91 (Academic Press, London, 2000).
[2】 Campbell, K.J. et al. Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques. Immunogenetics 61, 177-187 (2009).
[3]GouIder, P.J.R. & Watkins, D.I. Impact of MHC class I
diversity on immune control of immunodeficiency virus replication.
Nat. Rev. Immunol. 8,619-630 (2008)
[4]0'Leary, C.E. et al. Identification of novel MHC class I
sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing. Immunogenetics 61, 689-701
(2009).
[5] Robinson J, Malik A, Parham P, Bodmer JG, Marsh
SGE.IMGT/HLA database-a sequence database for the human major
histocompatibility complex. Tissue Antigens 55, 80-7 ( 2000 ) .
[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. the content of the invention by second generation sequencing technologies in order to be used for HLA-C Genotypings, the invention provides 3 pairs of PCR primers of 2,3 and 4 exons for expanding HLA-C genes, and they are the SEQ ID NO shown in table 1 respectively:1 and 2,3 and 4 and 5 and 6.3 pairs of PCR primers have good conservative and specificity, and can cover HLA-C sites 2,3 and 4 exon full length sequences, and PCR primer length is respectively less than 700bp, meet normal Illumina Solexa sequencings and require.In addition, the primer of the present invention applies also for the sequencing of Sanger methods.
The PCR primer of the HLA-C genes 2,3 of table 1. and 4 exons:
Present invention also offers a kind of new HLA-C genes 2,3 and 4 exon amplification methods, it is characterised in that is expanded using the amplimer of the present invention to entering performing PCR, the sequence of the amplimer pair is shown in table 1.
HLA-C 2,3 and 4 exons are amplified due to that can be reacted by PCR, therefore, method of the invention particularly advantageously can be used for carrying out HLA-C Genotypings.Compared with existing HLA-C methods of genotyping, due to being controlled in using the method for the present invention and the product of amplimer within 700 bp, therefore when further carrying out parting, it is possible to use be based on
The HLA-SBT of Illumina Solexa sequencing technologies.
Present invention also offers a kind of method that HLA-C genes 2,3 and/or 4 exons in sample is sequenced, it includes step：
1) provide a sample and extract the DNA of the sample;
2) primer pair of the present invention is used to expand the DNA to obtain PCR primer,
Preferred pair PCR primer is purified；
3) PCR primer is sequenced, the sequencing is preferably through second generation PCR sequencing PCR, and the second generation sequence measurement is such as Illumina Solexa or Roche454.
Present invention also offers a kind of HLA-C methods of genotyping, methods described includes：
1) enter performing PCR using the primer pair of the present invention to expand, expand the HLA-C genes 2,3 and/or 4 exons of sample to be tested；
2) extron amplified is sequenced, and be compared sequencing result with the standard sequence in database, so that it is determined that genotypic results, wherein described sequencing is by Sanger PCR sequencing PCRs, either by second generation PCR sequencing PCR, the second generation sequence measurement is for example
Illumina Solexa or Roche454.
On the other hand, present invention also offers a kind of kit for being used to carry out HLA-C Genotypings, the kit includes the pcr amplification primer thing pair of the present invention.In one embodiment, the kit also includes other reagents, such as reagent for DNA cloning, DNA purifying and/or DNA sequencings.
The amplimer pair and methods of genotyping provided using the present invention, can be in amplification
Genotyping is carried out on the basis of HLA-C 2,3 and 4 exons.Therefore in terms of existing technologies, the parting make use of Illumina Solexa sequencing technologies, improves flux, simplifies flow, while also saving time and cost.Illustrate the HLA-C 2 that Fig. 1 is part sample, 3,4 exon PCR primer electrophoresis results, from electrophoretogram, PCR primer clip size is respectively the single band being located between 400-500 bp, and wherein swimming lane M is DNA standard molecular weight objects of reference（DL 2000, Takara company）.
Fig. 2 shows the situation that rear DNA running gels rubber tapping is interrupted to HLA-Mix, and rubber tapping region is 450-750 bp regions.Wherein swimming lane M is molecular weight standard thing（NEB-50bp DNA Ladder), swimming lane 1 shows the glue figure of HLA-Mix before tapping rubber, and swimming lane 2 shows the glue figure of HLA-Mix after rubber tapping.
Fig. 3 shows the 2 exon consensus sequence construction procedures sectional drawings in the HLA-C sites of No. 2 samples.First, the sequencing sequence in the C sites of the sample is compared onto reference sequences by BWA softwares, the consensus sequence of the exon of sample C sites 2,2,4 is constructed respectively, just the blunt linkage relationship according between SNP determines the haplotype sequence of each extron in C sites again, finally, the type of the sample is determined by the common factor of each extron haplotype sequence.As illustrated, in No. 2 sample C
2 heterozygosis SNP are contained in the region of gene order 695-764, can determine that SNP linkage relationships are respectively by readl and read2：(" ... " in figure represents and reference sequences identical base A-C, G- A）.Its sequence corresponds to the dash area of C*010201 and C*07020101 type sequences respectively.The judgement of the linkage relationship in other regions is similar.
Fig. 4 shows 26 parts of sample HLA-C sites 2,3 and 4 exon PCR primer electrophoretograms.
26 parts of sample HLA-C sites 2,3 and 4 exon PCR primer electrophoretograms, as shown in FIG., all PCR primers are respectively less than 500 bp, and electrophoretic band is single, it is homogeneous in the amplification efficiency of each sample with a pair of primer pairs without obvious non-specific band.
Result C*08 is shown in No. 1 template PCR primer sequencing result of the peak figure through uType software analysis of Fig. 5 displays amplification, left side result Output bar:01:01 C*15:05:01, it is identical with the former known type of No. 1 template.Embodiment proposes following examples, and to make those of ordinary skill in the art more fully understand the present invention, the embodiment is not intended to limit the scope of the present disclosure merely for exemplary purpose.
The present invention adopts 3 pairs of PCR primers of design amplification HLA-C 2,3 and 4 exons with the following method.All newest HLA-C gene orders are downloaded from IMGT/HLA internet sites, are then saved in local disk as HLA-C data sets；All newest non-HLA-C HLA-I genoids sequence is downloaded simultaneously as comparing data set.Two data sets are compared, guarded and distinguished sequence at 2,3 and 4 exon two ends and internal each gene loci of searching, and the PCR primer sequence of design and mankind's whole genome sequence are subjected to tetraploid rice.Because HLA-C genes and the other genes for belonging to HLA-I quasi-molecules have very high sequence similarity, guarantee primer 3 of being tried one's best when designing PCR primer, end is special, it is ensured that primer is expanded
The specificity of HLA-C genes.The length of PCR primer is set to be less than 700 bp simultaneously, and the annealing temperature of positive anti-primer is consistent substantially.The multipair candidate HLA-C primers for meeting design requirement are used to expanding a small number of template DNAs with HLA-C common serotypes, conservative is therefrom filtered out and specificity is best, be respectively used for amplifying HLA- (：2,3 and 4 exons 3 pairs of PCR primers.
The present invention states method can be with
Method, those skilled in the art can carry out according to the conventional method, or be carried out according to the operation instructions of sequencing instrument. '
For example, in sequencing procedure is carried out using two generation sequencing technologies, 5, end addition primer label can be used（Primer index) sequence Tag primer carry out, PCR products after amplification can be entered Break Row, and interrupt rear product carry out end reparation and its 3, end connection desoxyadenossine (A) then connects different PCR-free joints.
It is that multiple samples are sequenced simultaneously in order to realize that one section of sequence label is connected in amplimer front end.Specifically, PCR-index/barcode technologies can be combined, by the way that the 5 of PCR primer, addition primer label (primer index) sequent synthesis Tag primer in end introduces unique primer label during PCR to each sample.So, in using second generation DNA sequencing technology detection process, except PCR links must be one by one in addition to sample process, other experiment links can mix multiple samples and handle simultaneously, and the testing result of final each sample can be given for change by its unique primer label sequence.The design of primer label is different and different according to the experiment porch applied, it is considered to the characteristics of Illumina GA microarray datasets itself, the present invention mainly considered in design primer label it is following some： 1:More than 3 are avoided in primer label sequence（Including 3）Single base repetitive sequence, 2:Base A and base C total content are accounted between the 30%-70% of all base contentses in the same site of all primer labels, and 3:The G/C content of primer label sequence in itself exists
Between 40-60%, 4:Sequence difference degree is more than 4 bases, 5 between primer label:The sequence high with Illumina GA sequencing primer similarities, 6 are avoided the occurrence of in primer label sequence:After reduction primer label sequence is added in PCR primer, the serious hair fastener caused to PCR primer（Hairpin), dimer（Dimer) the appearance of situation.
Term " PCR-Free libraries joint（Adapter one section of base through design) " is referred to; its main conjunction site; PCR-Free libraries joint can be connected directly to the DNA fragmentation two ends in sequencing library by DNA ligase; the importing process of joint is because the participation without PCR, therefore referred to as PCR-Free libraries joint." joint（), or " library joint adapter（Library adapter), label technique refers to by adding different library joints to multiple sequencing libraries（The composition sequence of different library joints is different, and the different part of sequence is referred to as splice tag（Adapter index)), label sequencing library is built, so that multiple different label sequencing library mixing sequencings can be realized, and a kind of library label technique that finally sequencing result in each label sequencing library can be distinguished mutually.For example, coming from ILLUMIA using PCR-FREE libraries joint in the embodiment of the present invention.
In examples below, with the 3 pairs of PCR primers filtered out, the blood sample of HLA Common genes type known to 95 carries out HLA-C site PCR amplifications, and amplified production is sequenced through Sanger methods and second generation sequence measurement.Sequencing result is used for HLA-C partings, and verifies by being compared with former genotyping result the conservative and specificity of PCR primer.
Embodiment 1. carries out the extraction of the sample DNA of HLA-C Genotypings 1. with second generation sequencing technologies Il miina GA
Extracted using KingFisher automatic extracting instruments in 95 known other blood samples of HLA genotype and extract DNA.Key step is as follows：The supporting deep-well plates of 6 Kingfisher automatic extracting instruments and 1 shallow bore hole plate are taken out, a certain amount of supporting reagent and mark are separately added into according to specification, has added the orifice plate of good reagent to be placed in corresponding position, selection procedure on request by all
" the Blood DNA of the μ of Bioeasy-200 1-KF.msz " program, presses " start " and performs program progress nucleic acid extraction.The eluted product that 100 μ in plate Elution or so are collected after EP (end of program) is the DNA of extraction, is used as the template in next step PCR.
2. PCR is expanded
By synthesis 5, there is the PCR primer of different primers label to make different PCR Tag primers for end, and these different PCR Tag primers can be used for different samples, and the PCR primers are 2, the 3 and 4 exon PCR primers for HLA-C.Primer label is introduced at PCR primer two ends thereafter by PCR reactions, so that PCR primer of the specific marker from different samples.
Expand 95 parts of DNA samples respectively with 95 sets of PCR Tag primers, often cover PCR primer of the PCR Tag primers by 2,3 and 4 exons for expanding HLA-C（Table 1) and a pair of two-way primer labels（Table 2) composition, wherein the 5 of each forward direction PCR primer, connect the forward primer label of pair of primers label on end, and the 5 of inverse PCR primer, the reverse primer label of pair of primers label is connected on end.Primer label is directly added in the 5 of PCR primer, end when primer is synthesized.
95 parts of DNA of gained in sample extraction step, number consecutively 1-95, PCR reaction is carried out in 96 orifice plates, totally 3 plate, and numbering is respectively HLA-P-C2, HLA-P-C3, and (C2/3/4 represents the site of amplification to HLA-P-C4）, plate is interior to set a negative control without template, and negative control the primer is identical with primer PI-96.While experiment, the corresponding sample number information of each pair primer label is recorded.
The relevant information of primer label:
The orifice plate position corresponding templates of numbering Direct/Reverse 96
PI-1 TCGCAGACATCA TGACACGATGCT A01 1
PI-2 TACATCGCACTA TACAGATGCTGA A02 2
PI-3 CTCGATGAGTAC ACGTCTAGACAC A03 3
PI-4 TCTGTATACTCA TGCTGTAGTGAC A04 4
PI-5 TATCTGCTCATA AGATATCGAGCT A05 5
PI-6 TACATGCTGAGC ACGTGTCTATCA A06 6
PI-7 TCATATCGCGAT AGATCGTATAGC A07 7
PI-8 ACAGATGCACGC ATCTCGTGACAG A08 8
PI-9 TAGATCGTACAT ACTAGTACACGC A09 9
PI-10 ACTACACGTCTC ATAGTCACGCGT A10 10
PI-11 AGACTCGCGTAT TACTAGCTGACG All 11
PI-12 ATACTAGTGCTC TGTATCGTGCTC A12 12
PI-13 CACGATGACATC TAGTGAGCGCAC B01 13
PI-14 TGCTGTCTCGAG CATAGCAGTGTC B02 14
PI-15 TGTGCTCGAGTC TCTGATCGAGCA B03 15
PI-16 CACTCGTACATC AGCGATGCTCAT B04 16
PI-17 CGACGTGCTCGC CGCGTACTGCAG B05 17
PI-18 ACGCATCTATAC CTAGTATCGCAG B06 18
PI-19 CGAGATGACTCT TGTATACACGAT B07 19
PI-20 ACTGTCTCGAGC ACGTAGCGCACA B08 20
PI-21 CATCTGCTATAG TCTAGCTCATGA B09 21
PI-22 ACGCACTCTAGA CTATGCACTGAT BIO 22
PI-23 TGAGATACAGTA ATCTGCTATGAC Bll 23
PI-24 ACTCATCGTGCT TAGAGCTGTCAC B12 24
PI-25 TACACTGTCTAT CAGCACATAGAT C01 25
PI-26 CACAGTACTCGC CTGCTAGTGTAT C02 26
PI-27 TGTACTATCATA TGTGATAGACAC C03 27
PI-28 CTAGTACTGACG AGCGAGTCTACT C04 28
PI-29 TAGACTGAGCTA ACATACTGAGAC C05 29
PI-30 CAGACGCGTGAG TACATCTCGTAT C06 30
PI-31 CGCGACATCACG TAGCGATGAGAC C07 31
PI-32 ACACTCATAGAT CTATCATGACAC C08 32
PI-33 AGCGTATACTAG CATACTCACGTA C09 33
PI-34 TGTCGTGCTATC ACATGACTCACG CIO 34
PI-35 CGCTAGACTGTA TACTATAGTCGA Cll 35
PI-36 ACAGTGTAGCGC TGATATGCTACA C12 36
PI-37 CACTCTATCGAC TCACGCGATGAG D01 37
PI-38 ACACTCTAGTCA ACGTAGATCTAT D02 38
PI-39 CATATGAGATCG AGCAGAGTGCTC D03 39
PI-40 CAGCTATCATAC CACTGCAGACGA D04 40
PI-41 TATACTCTAGAT TGCATAGAGCGC D05 41
PI-42 TGTATGCTCGTC TCGTGACAGATC D06 42
PI-43 TAGTGATGCTCT ACGAGCTGATAT D07 43
PI-44 AGACTCTGAGTC CTGATAGTATCA D08 44
PI-45 CTCATAGACTAC ATCGCGAGTGAC D09 45
PI-46 TCGCTCACTACA TGTCTCGACATC D10 46
PI-47 ATAGAGTCTCAT CGCATAGCGTAT Dll 47
PI-48 CGAGACACTCGC TCGTAGTCTACA D12 48
PI-49 CAGCATACTATC TCGTGATACAGA E01 49
PI-50 CAGCTATAGTCT ATGCAGATATCT E02 50
PI-51 TCTATCGATGCA ACACGCAGATCG E03 51
PI-52 CATGAGTATAGC CTAGCTGACGTA E04 52
PI-53 TAGCATATCGAG TACACGTATGAG E05 53
PI-54 ACGACTCGCTAC TCATGACTAGTA E06 54
PI-55 TAGCATACACGC TGACGCGTATAC E07 55
PI-56 CGTCATATGCAG TATAGCGATGAC E08 56
PI-57 TGCAGCGAGTAC TCGACGCTAGCG E09 57
PI-58 CGTGTCGACAGA CAGTCGTGAGCA E10 58
PI-59 ACTCGACGTGAG ACGCGAGTGATA Ell 59
PI-60 ACTCGTCTGACG TGCTATCACTGA E12 60
PI-61 CATACTGTATCT TACATAGATGTC F01 61
PI-62 TCTACTCGTGAC CACGTATAGTGA F02 62
PI-63 CTGCACTAGACA ACTCATATCGCA F03 63
PI-64 ACACGAGCTCAT CACTCATATCGA F04 64
PI-65 TACAGATAGTCT TCGTCTGTGATA F05 65
PI-66 TACACTCGTGCT TGACGCTCATCT Γ06 66
PI-67 TACATGTGACGA TCGTACATGCTC F07 67
PI-68 TGTATGATCTCG CACTGTGCTCAT F08 68
PI-69 CAGTACACTCTA ACTGCATGATCG F09 69
PI-70 CATACTATCACG TCGTGTCACTAC F10 70
PI-71 CACTATACAGAT CGACACGTACTA Fll 71
PI-72 ATATCGTAGCAT TCGTGATCACTA F12 72
PI-73 TAGTCTATACAT AGACGCTGTCGA G01 73
PI-74 TGTCACAGTGAC TCATATGATCGA G02 74
PI-75 ATCGACTATGCT CGATCATATGAG G03 75
PI-76 ATACTAGCATCA TCATGCTGACGA G04 76
PI-77 CACTGACGCTCA CACTACATCGCT G05 77
PI-78 TCGCTCATCTAT TAGTACAGAGCT G06 78
PI-79 TGTATCACGAGC ATGATCGTATAC G07 79
PI-80 TACTGCTATCTC CGCTGCATAGCG G08 80
PI-81 CGCGAGCTCGTC ACTCGATGAGCT G09 81
PI-82 TAGAGTCTGTAT TGTCTATCACAT G10 82
PI-83 TACTATCGCTCT TATGTGACATAC Gil 83
PI-84 TAGATGACGCTC TACTCGTAGCGC G12 84
PI-85 TCGCGTGACATC ATCTACTGACGT HOI 85
PI-86 ACACGCTCTACT ACAGTAGCGCAC H02 86
PI-87 TACATAGTCTCG CTAGTATCATGA H03 87
PI-88 TGAGTAGCACGC TCGATCATGCAG H04 88
PI-89 TAGATGCTATAC TACATGCACTCA H05 89
PI-90 ATCGATGTCACG CAGCTCGACTAC H06 90
PI-91 ATCATATGTAGC CTCTACAGTCAC H07 91
PI-92 TAGCATCGATAT AGATAGCACATC H08 92
PI-93 TGATCGACGCTC CTAGATATCGTC H09 93
PI-94 TGCAGCTCATAG TACAGACTGCAC H10 94
PI-95 CGACGTAGAGTC CAGTAGCACTAC Hll 95
PI-96 CACTGTATAGCT CGACTAGTACTA H12 negative controls are by the DNA in step 1 using the extraction of KingFisher automatic extracting instruments as template, and with 5, HLA-C each extron primer one tube PCR amplification of the end with label, PCR programs are as follows： C2:96 " C 2 minutes；95 °C 30s 62 °C 30 seconds 72 °C 20 seconds（35 circulations）； 15 °C oo ;
C3:96 °C 2 minutes；95 °C 30 seconds 56 °C 30 seconds 72 °C 20 seconds（35 circulations）； 15°C∞;
C4:96 °C 2 minutes；95 °C 30 seconds 60 °C 30 seconds 72 °C 20 seconds（35 circulations）； 15°C∞.
HLA-C PCR reaction systems are as follows：
Wherein PInrC-F2/3/4Represent primer 5, HLA-C of the end with No. n-th forward primer sequence label (table 2) F primers, PInrC-R2/3/4Represent primer 5, HLA-C of the end with No. n-th reverse primer sequence label R primers（N≤96) herein, other the like.And each specific a set of PCR primer of sample correspondence.
PCR reactions are run in the PTC-200 PCR instruments of Bio-Rad companies.After the completion of PCR, 2 l PCR primers are taken to be detected through 1.5% agarose gel electrophoresis.Fig. 1 shows the corresponding extron PCR primer electrophoresis results of preceding 20 sample HLA-C, and DNA molecular marker is (the Takara companies of DL 2000）, Jiao Tushang have a series of clip sizes be the 400 single bands of bp-500 bp, show each extrons of HLA-C of this part sample（C2, C3, C4) PCR expands successfully.The result of other samples is similar.
3. PCR primer is mixed and purified
From the 96 remaining PCR primers of orifice plate HLA-P-C2（Except negative control）Respectively take
20 μ are blended in 3 ml Ε Ρ pipes, labeled as HLA-C2-Mix, other 2 96 orifice plates are carried out with same operation, it is respectively labeled as HLA-C3-Mix and HLA-C4-Mix, concussion is mixed, 200 μ are respectively taken to be blended in 1.5 ml Ε Ρ pipes from HLA-C2-Mix, HLA-C3-Mix and HLA-C4-Mix, labeled as HLA-Mix, 500 μ DNA mixtures are taken from HLA-Mix through Qiagen DNA Purification kit (QIAGE companies）Cross post purifying（Specific purification step refers to specification）, the Ν Α of 200 μ 1 obtained by purifying, through (the Thermo Fisher Scientific companies of Nanodrop 8000）It is 50 ng l to determine HLA-Mix DNA concentrations.
4. the structure of Illumina GA PCR-Free sequencing libraries
4.1 PCR primers are interrupted
The μ of total amount 5 is taken from HLA-Mix after purification8DNA Covaris
MicroTube with AFA fiber and Snap-Cap are interrupted on Covaris S2 (Covaris companies).Interrupt condition as follows：
Frequency scan (frequency sweeping)
4.2 interrupt after PCR primer purifying
All by HLA-Mix interrupt product QIAquick PCR Purification Kit recovery purifyings, are dissolved in respectively in 37.5 μ EB (QIAGEN Elution Buffer).
Repair reaction in 4.3 ends
The reparation reaction of DNA ends is carried out to the product of purifying, system is as follows（Reagent is purchased from
Enzymatics companies)：
Reaction condition is：Under 20 °C, in Thermomixer (Eppendorf companies）Middle warm bath 30 minutes.
Reaction product is dissolved in 32 μ EB (QIAGEN Elution Buffer) through QIAqukk PCR Purification Kit recovery purifyings.
4.4 3, end adds A to react
Previous step reclaims the 3 of DNA, and end adds A to react, and system is as follows（Reagent is purchased from Enzymatics companies)：
The μ reaction conditions of Klenow (3'-5'exo-) 3 μ cumulative volumes 50 are：Under 37 °C, warm bath 30 minutes in Thermomixer.
Reaction product is through MiniElute PCR Purification Kit (QIAGE companies）Pureization is reclaimed, 38 μ Ε Β solution is dissolved in（QIAGEN Elution Buffer) in.
4.5 connection Illumina GA PCR-Free libraries joints（adaptor )
Plus the product after A connects Illumina GA PCR-Free libraries joint, system is as follows（Reagent is purchased from Illumina companies）：
Reaction condition is：Under 16 °C, warm bath is stayed overnight in Thermomixer.
Reaction product is dissolved in 50 μ deionized waters after purification through Ampure Beads (Beckman Coulter Genomics), and it is as follows to detect DNA concentration result through quantitative fluorescent PCR (QPCR)：
4.6 rubber tapping are reclaimed
The HLA-Mix of 30 μ 1 are taken to be reclaimed with 2% low melting-point agarose glue.Deposition condition is 100V, 100 minutes.DNA standard molecular weights object of reference is 50 bp DNA Ladder of NEB companies.The DNA fragmentation of 400-750 bp length ranges is reclaimed in rubber tapping（Fig. 2).Glue reclaim product is through QIAquick PGR Purification Kit (QIAGEN companies）Recovery purifying, volume is 32 μ after purification, and DNA concentration result is detected for 17.16 nM through quantitative fluorescent PCR (QPCR).
5. Illumina GA are sequenced
According to QPCR testing results, 10 pmol DNA are taken to be sequenced with Illumina GA PE-100 programs, concrete operations flow refers to Illumina GA operational manuals（Illumina GA II x ) .
6. interpretation of result
The sequencing result of Illumina GA outputs is a series of DNA sequence dnas, is sequenced by searching
As a result positive and negative primer label sequence and primer sequence in, set up each primer label correspondence sample
The database of each extron PCR primer sequencing results of HLA-C；Pass through BWA
(Burrows- Wheeler Aligner) is positioned at the sequencing result of each extron on the reference sequences of corresponding extron（Reference sequences are originated： http：〃 www.ebi.ac.uk/imgt/hla/), and build the uniformity of each database（Consensus) sequence；With reference to base sequencing quality value and sequencing sequence and the diversity factor of consensus sequences, error correction is screened and is sequenced to sequencing sequence；And the DNA sequence dna after correction passes through overlapping sequences（Overlap it is) and chain
(Pair-End is chain）Relation can be assembled into the corresponding sequence of each extrons of HLA-C.Fig. 3 example screenshot illustrates the process built to the 2 exon consensus sequences in the HLA-C sites of No. 1 sample.
The sequence library of the DNA sequence dna for the HLA-C intrones being sequenced each extron corresponding to HLA-C in IMGT HLA specialized databases is compared, what sequence alignment result 100% was matched is the HLA-C gene types of correspondence sample.All 95 samples, obtained genotyping result is consistent completely with former known genotyping result, and the wherein concrete outcome of 1-32 samples and sample original genotyping result contrast is as follows：（As shown in table 3, whole testing results are identical with original testing result）.
This genotyping result of table 3. is contrasted with the original genotyping result of sample:
21 C*03:03 C*08:01 C*03:03 C*08:01 is
22 C*03:04 C*07:02 C*03:04 C*07:02 is
23 C*07:02 C*08:01 C*07:02 C*08:01 is
24 C*07:02 C*12:02 C*07:02 C*12:02 is
25 C*07:02 C*12:03 C*07:02 C*12:03 is
26 C*03:04 C*08:01 C*03:04 C*08:01 is
27 C*01:02 C*03:04 C*01:02 C*03:04 is
28 C*07:02 C*12:02 C*07:02 C*12:02 is
29 C*03:02 C*07:02 C*03:02 C*07:02 is
30 C*01:02 C*03:03 C*01:02 C*03:03 is
31 C*01:02 C*07:02 C*01:02 C*07:02 is
32 C*01:02 C*07:02 C*01:02 C*07:02 is note：C*0303 in HLA-C types is not excluded for C*0320N possibility, C*0401 is not excluded for C*0409N/0430 possibility, C*0702 is not excluded for C*0750 possibility, C*0801 is not excluded for C*0822 possibility, C*1505 is not excluded for C*1529 possibility, because above-mentioned allele is identical in the sequence of the exons of HLA-C 2,3,4.
Embodiment 2. carries out HLA-C Genotypings with the sequencing of Sanger methods
1. sample DNA is extracted
It is similar as described in example 1 above, 26 known other DNA of HLA genotype in 95 samples extracted with KingFisher automatic extracting instruments.
2. PCR is expanded
Using above-mentioned KingFisher automatic extracting instruments extract DNA as template, with
Totally 3 pairs of PCR primers distinguish one tube PCR amplification by C-F2, C-R2, C-F3, C-R3, C-F4, C-R4, and each pair of primer PCR program is as follows：
C2:96 " C 2 minutes;95 " C 30 seconds 62 °C 30 seconds -72 °C 20 seconds（35 circulations）； 15 °C oo ;
C3:96 °C 2 minutes;95 " 30 seconds-^56 of C " C 30 seconds 72.C (35 circulations in 20 seconds）； 15°C oo ;
C4:96 °C 2 minutes;95 " C 30 seconds 60 °C 30 seconds 72 °C 20 seconds（35 circulations）； 15 °C∞.
HLA-C PCR reaction systems are as follows：
DNA (about 20 ng/ μ) 2.0 μ
ddH20 12.8 μΐ
Amount to 25.0 μ
After PCR primer is detected through agarose gel electrophoresis（Fig. 4), purifying is prepared.
3. PCR primer is purified
PCR primer purifying is carried out using millipore purifying plates.Basic step is：Purify the hole for marking on plate and needing to use in 96 hole PCR primers with marking pen, and the ultra-pure waters of 50 μ 1 are added into the hole needed to use, remaining hole pastes sealed membrane, it is stored at room temperature 15 minutes or is connected in suction filtration system, -10 Pa, remove within 5 minutes, the liquid for remaining in purifying plate bottom leakage fluid dram will be blotted when removing purifying plate from suction filtration system every time on blotting paper.
PCR primer centrifugation to be purified, 4000 rpm, 1 minute；Open in the lid or silicagel pad of PCR primer to be purified, each PCR reaction systems and add 100 μ ultra-pure waters.Then the purifying plate for adding PCR primer to be purified is connected in suction filtration system, regulation vacuum to air gauge shows no liquid on -10 handkerchiefs, suction filtration to the micropore regenerated fiber film for purifying plate bottom, is observed under illumination, without complete liquid level specular gloss.
To needing the μ ultra-pure waters of Kong Zhongjia 50 or Τ Ε of purified pcr product to micropore regenerated fiber film；Plate is purified using frequency modulated oscillations in micro oscillator 5 minutes, whole liquid are into the new corresponding hole of 96 hole PCR plates in transfer respective aperture at room temperature.
4. carry out sequencing reaction and purify sequencing reaction product
Sequencing reaction is done by template of above-mentioned PCR primer after purification, the condition of sequencing reaction is：96 °C 2 minutes；96 °C 10 seconds-> 55.C 5 seconds 60 °C 2 minutes（25 circulations）； 15 "C ∞.
The system of sequencing reaction is：
Sequencing reaction product is purified by following steps：Sequencing reaction plate trim is removed, is centrifuged 1 minute under 3000 g.The every 5 μ reaction systems of 96 orifice plates add the mol/L EDTA-Na2 solution of 2 μ 0.125, and the ethanol of 33 μ 85% covers silicagel pad, fully vibration 3 minutes, under 4 °C with
3000 g are centrifuged 30 minutes.Sequencing plate is taken out in centrifugation after terminating, open silicagel pad, sequencing reaction plate is inverted on blotting paper, the heterogeneous example showing an absence of inverse disconnection between the middle term and the major term heart to centrifugal force reaches to be stopped immediately during 185 g.96 orifice plates add the ethanol of 50 μ 70% per hole, cover silicagel pad, vibrate 1.5 minutes, are centrifuged 15 minutes with 3000 g under 4 °C.Sequencing reaction plate puts lucifuge ventilation 30 minutes, air-dries to without ethanol smell.96 orifice plates add 10 μ (384 orifice plates add 8 μ per hole) HI-DI Yue acid amides per hole, and lid sealed membrane, vibration is centrifuged to 1000 rpm after 5 seconds.
5. sequencing and interpretation of result
Sequencing reaction product after purification carries out Capillary Electrophoresis order-checking on ABI 3730XL, and sequencing peak figure is analyzed by uType softwares (Invitrogen)（Fig. 5), HLA genotyping results are obtained.Whole testing results are identical with original testing result（Table 4).
This genotyping result of table 4. is contrasted with the original genotyping result of sample：
It will be apparent to one skilled in the art that without departing substantially from the scope of the present invention and essence
On the premise of god various modifications and changes can be carried out to the present invention.By considering specification disclosed herein of the invention and example, other embodiments of the invention are apparent to those skilled in the art.This specification and embodiment should be regarded solely as example of use, and really scope and spirit illustrate the present invention in the appended claims.
Bibliography=
[1]. http://www.ebi.ac.uk/imgt/hla/stats.html
[2]. Tiercy J M. Molecular basis of HLA polymorphism:
implications in clinical transplantation. Transpl Immunol, 2002, 9:
173-180.
[3] Wegner KM: Massive parallel MHC genotyping: titanium that shines. Molecular Ecology 2009, 18: 1818-1820.
[4] Ellegren H:The and takes large-scale genomics into the wild. Molecular Ecology 2008,17 of Sequencing goes 454: 1629-1635.
[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
transplantation 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.
Claims (10)
- Claims1. with SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO:5 and SEQ ID NO:The polynucleotides of sequence shown in 6 any one.2. a kind of HLA-C genes 2,3 and/or 4 exon amplification methods, it is characterised in that using selected from 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 primer pair enters performing PCR amplification, amplification HLA-C genes 2,3 and/or 4 exons.3. a kind of method that HLA-C genes 2,3 in sample and/or 4 exons are sequenced, it includes step：1) provide a sample and extract the DNA of the sample;2) SEQ ID NO will be selected from: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 primer pair is used to expand the DNA to obtain PCR primer, and preferred pair PCR primer is purified；3) PCR primer is sequenced.4. the method for claim 3, wherein the sample is blood sample.5. the method for claim 4, wherein the blood sample comes from mammal or people.6. a kind of HLA-C methods of genotyping, methods described includes：1) using selected from 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 primer pair is carried outPCR is expanded, and expands the HLA-C genes 2,3 and/or 4 exons of sample to be tested；2) extron amplified is sequenced, and sequencing result is compared with the standard sequence in database, so that it is determined that genotypic results.7. the method for claim 3 or 6, wherein the sequencing is by Sanger PCR sequencing PCRs, or passes through second generation PCR sequencing PCR.8. the method for claim 7, wherein the second generation sequence measurement is Illumina Solexa or Roche454.9. a kind of kit for being used to carry out HLA-C Genotypings, the kit includes SEQ ID NO:1 and SEQ ID NO: 2; SEQ ID NO:3 and SEQ ID NO: 4;And/or SEQ ID NO:5 and SEQ ID NO:6 pcr amplification primer thing pair, the kit preferably also includes the reagent for DNA cloning, DNA Pureization and/or DNA sequencing.10. the method for the polynucleotides of claim 1, claim 6 or the kit of claim 9 are used for the purposes of HLA-C Genotypings.
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| CN109371114A (en) * | 2018-12-26 | 2019-02-22 | 银丰基因科技有限公司 | HLA-DQB1 genotyping kit |
| CN110621785A (en) * | 2017-06-20 | 2019-12-27 | 深圳华大生命科学研究院 | Method and device for typing diploid genome haploid based on third generation capture sequencing |
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| US7300755B1 (en) * | 2003-05-12 | 2007-11-27 | Fred Hutchinson Cancer Research Center | Methods for haplotyping genomic DNA |
| US20100261189A1 (en) * | 2008-10-03 | 2010-10-14 | Roche Molecular Systems, Inc. | System and method for detection of HLA Variants |
| CN101921842A (en) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof |
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| CN101921841B (en) * | 2010-06-30 | 2014-03-12 | 深圳华大基因科技有限公司 | HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on Illumina GA sequencing technology |
| CN101921840B (en) * | 2010-06-30 | 2014-06-25 | 深圳华大基因科技有限公司 | DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method |
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| US7300755B1 (en) * | 2003-05-12 | 2007-11-27 | Fred Hutchinson Cancer Research Center | Methods for haplotyping genomic DNA |
| US20100261189A1 (en) * | 2008-10-03 | 2010-10-14 | Roche Molecular Systems, Inc. | System and method for detection of HLA Variants |
| CN101921842A (en) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | HLA (Human Leukocyte Antigen)-A,B genotyping PCR (Polymerase Chain Reaction) primer and application method thereof |
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| CN110621785A (en) * | 2017-06-20 | 2019-12-27 | 深圳华大生命科学研究院 | Method and device for typing diploid genome haploid based on third generation capture sequencing |
| CN110621785B (en) * | 2017-06-20 | 2023-08-15 | 深圳华大生命科学研究院 | Method and device for haplotyping diploid genome based on three-generation capture sequencing |
| CN109371114A (en) * | 2018-12-26 | 2019-02-22 | 银丰基因科技有限公司 | HLA-DQB1 genotyping kit |
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