Background technology
Human leucocyte antigen (human leucocyte antigen, HLA) is the important component part of human immune system, is also the genetic polymorphism sexual system that known human body is the most complicated at present.According to the feature of the aspects such as its structure, tissue distribution and function, HLA molecule can be divided into three quasi-molecule such as HLA-I, HLA-II and HLA-III, wherein HLA-I quasi-molecule is main relevant to immunological rejection with HLA-II quasi-molecule function, and HLA-III quasi-molecule function is main relevant to the synthesis of Ia part complement system and inflammation-related factor etc.; Clinical transplantation research finds, the being proportionate property of matching degree of HLA-I and the HLA-II molecule of the long-term surviving rate of graft and donor and receptor both sides, and when the matching degree for the HLA molecule by both sides is higher, the long-term surviving rate of graft is higher.
Wherein HLA-C site belongs to classical HLAI genoid, and between HLA-A, B site, coding HLA-C molecule, its mrna length is about 3Kb, is made up of 7 introns and 8 exons.In recent years, along with the development of HLA genotyping technique, people find that HLA-C with HLA-A, B molecule are the same gradually, have height polymorphism, and confirm the importance of HLA-C molecule in clinical transplantation.
The HLA typing method of current international standard comprises PCR-SSP (sequence specific primers polymerase chain reaction), PCR-SSO (polymerase chain reaction oligonucleotide probe hybridization) and PCR-SBT (polymerase chain reaction product sequencing based type).
The principle of HLA-SSP designs a whole set of allelotrope group-specific primers, obtains the special amplified production of HLA type, determine HLA type by electrophoretic analysis by round pcr.The principle of HLA-SSO designs the special oligonucleotide sequence of HLA type as probe, PCR primer marked, with PCR primer (gene DNA to be detected) and probe hybridization.HLA type is judged by detecting fluorescent signal.The detection signal of HLA-SSP and HLA-SSO is all simulating signals, low-level during resolving power generally can only arrive and all can not detect new allelotrope.
HLA-SBT based on the order-checking of Sanger method is a kind of by directly carrying out Sanger method order-checking (kapillary Ventral medulla) to the DNA product after pcr amplification, measure nucleotide sequence, thus judging other high resolution classifying method of HLA genotype, it has intuitively, high resolution and can detect new allelic feature.But the shortcoming such as complicated based on the whole experiment flow of HLA-SBT of Sanger method order-checking, flux is low and experimental cost is high makes it be difficult to be applied to extensive HLA high resolution somatotype project.
HLA-SBT based on the s-generation sequencing technologies being representative with Illumina Solexa and Roche454 (hereinafter referred to as new sequencing technologies) is also a kind of by directly measuring nucleotide sequence to the DNA product after pcr amplification, thus judge other high resolution classifying method of HLA genotype, it is except original directly perceived, high resolution and can detect except the feature of neomorph, also has that single-molecule sequencing, experiment flow are simple, a feature of high-throughput and low cost.But compared with first-generation sequencing technologies (checking order the sequencing technologies based on principle by Sanger method), can be used for DNA length prepared by new sequencing technologies sequencing library can not oversize (the maximum prove-in length of current I llumina Solexa be 700bp), and new sequencing technologies is read long general partially short, current I llumiaGA is two-way to be read longly maximumly can reach 300bp.
In view of the feature of new sequencing technologies, the length of PCR primer is no more than 700bp, and the PCR primer of the former HLA-SBT based on Sanger method sequence measurement is no longer applicable.Therefore, the primer that a set of conservative property and the good and PCR primer length of specificity meet new sequencing technologies requirement is necessary to design.
Reference
[1]Marsh,S.G.E.,Parham,P.&Barber,L.D.The HLA Facts Book3–91(Academic Press,London,2000).
[2]Campbell,K.J.et al.Characterization of 47MHC class I sequences in Filipinocynomolgus macaques.Immunogenetics61,177–187(2009).
[3]Goulder,P.J.R.&Watkins,D.I.Impact of MHC class I diversity on immune control ofimmunodeficiency virus replication.Nat.Rev.Immunol.8,619–630(2008).
[4]O’Leary,C.E.et al.Identification of novel MHC class I sequences in pig-tailed macaquesby amplicon pyrosequencing and full-length cDNA cloning and sequencing.Immunogenetics61,689–701(2009).
[5]Robinson J,Malik A,Parham P,Bodmer JG,Marsh SGE.IMGT/HLA database-a sequencedatabase for the human major histocompatibility complex.Tissue Antigens55,80-7(2000).
[6]Elaine R.Mardis.The impact of next-generation sequencing 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 barcoding and pyrosequencing to identify rare HIV drug resistance mutations.Nucleic Acids Res.2007;35(13):e91.
Summary of the invention
In order to s-generation sequencing technologies is used for HLA-C gene type, the invention provides 3 pairs of PCR primer of 2,3 and 4 exons for the HLA-C gene that increases, they are the SEQ ID NO:1 and 2,3 and 4 and 5 and 6 of display in table 1 respectively.Described 3 pairs of PCR primer have good conservative property and specificity, and can cover HLA-C site 2,3 and 4 exon full length sequence, and PCR primer length is all less than 700bp, meet normal Illumina Solexa order-checking requirement.In addition, primer of the present invention is also applicable to the order-checking of sanger method.
The PCR primer of table 1.HLA-C gene 2,3 and 4 exon
Present invention also offers a kind of new HLA-C gene 2,3 and 4 exon amplification method, it is characterized in that using amplimer of the present invention to carrying out pcr amplification, the right sequence of described amplimer is shown in table 1.
Owing to can be amplified 2,3 and 4 exons of HLA-C by PCR reaction, therefore, method of the present invention particularly advantageously can be used for carrying out HLA-C gene type.Compared with existing HLA-C methods of genotyping, within being controlled in 700bp owing to using the product of method of the present invention and amplimer, therefore when carrying out somatotype further, the HLA-SBT based on Illumina Solexa sequencing technologies can be utilized.
Present invention also offers a kind of method checked order to HLA-C gene 2,3 and/or 4 exon in sample, it comprises step:
1) sample is provided and extracts the DNA of this sample;
2) primer pair of the present invention is used for increase described DNA thus obtain PCR primer, preferably purifying is carried out to PCR primer;
3) check order to described PCR primer, described order-checking is preferably through s-generation sequencing, and described s-generation sequence measurement is such as Illumina Solexa or Roche454.
Present invention also offers a kind of HLA-C methods of genotyping, described method comprises:
1) primer pair of the present invention is used to carry out pcr amplification, HLA-C gene 2,3 and/or 4 exon of amplification sample to be tested;
2) exon amplified is checked order, and the standard sequence in sequencing result and database is compared, thus determine genotypic results, wherein said order-checking is by Sanger sequencing, or by s-generation sequencing, described s-generation sequence measurement is such as Illumina Solexa or Roche454.
On the other hand, present invention also offers a kind of test kit for carrying out HLA-C gene type, described test kit comprises pcr amplification primer pair of the present invention.In one embodiment, described test kit also comprises other reagent, such as, for the reagent of DNA cloning, DNA purifying and/or DNA sequencing.
Apply amplimer provided by the invention to and methods of genotyping, can amplification HLA-C 2,3 and 4 exons basis on carry out gene type.Therefore in terms of existing technologies, this somatotype make use of Illumina Solexa sequencing technologies, improves flux, simplifies flow process, also saves time and cost simultaneously.
Embodiment
Propose following examples, to make those of ordinary skill in the art understand the present invention better, described embodiment, only for exemplary purpose, is not intended to limit the scope of the present disclosure.
The present invention adopts 3 pairs of PCR primer of 2,3 and 4 exons of following method design amplification HLA-C.Download all up-to-date HLA-C gene orders from IMGT/HLA internet site, be then saved in local disk as HLA-C data set; Download the HLA-I genoid sequence of all up-to-date non-HLA-C as comparative data collection simultaneously.Two data sets are compared, guards and distinguished sequence at 2,3 and 4 exon two ends and each gene locus of inner searching, and the PCR primer sequence of design and mankind's whole genome sequence are carried out tetraploid rice.Because HLA-C gene and other gene belonging to HLA-I quasi-molecule have very high sequence similarity, ensure that when designing PCR primer primer 3 ' end is special, guarantees the specificity of primer amplification HLA-C gene as far as possible.Make the length of PCR primer be less than 700bp, and the annealing temperature of positive anti-primer is consistent substantially simultaneously.The minority that is used for by the multipair candidate HLA-C primer met design requirement increasing has the template DNA of HLA-C common serotype, therefrom filters out conservative property and specificity is best, 3 pairs of PCR primer of 2,3 and 4 exons of the HLA-C that is respectively used to increase.
The method of the DNA cloning method related in the present invention, method from sample extraction DNA, DNA purification process and DNA sequence dna comparison can be any methods availalbe this area.Described method can be selected as the case may be by those skilled in the art.For the method for DNA sequencing, those skilled in the art can carry out according to the method for routine, or carry out according to the working instructions of order-checking instrument.
Such as, utilize two generation sequencing technologies carry out in sequencing procedure, the Tag primer that 5 ' end can be used to add primer label (primerindex) sequence carries out, PCR primer after amplification can be interrupted, and interrupt after product carry out end reparation and connect Desoxyadenosine (A) at its 3 ' end, then connect different PCR-free joints.
Connect one section of sequence label in amplimer front end be check order to multiple sample to realize simultaneously.Specifically, in conjunction with PCR-index/barcode technology, primer label (primer index) sequent synthesis Tag primer can be added by the 5 ' end in PCR primer, in PCR process, unique primer label introduced to each sample.Like this, utilizing in s-generation DNA sequencing technology for detection process, except PCR link must one by one except sample process, other experiment link can mix multiple sample and process simultaneously, and the detected result of final each sample can be given for change by the primer label sequence of its uniqueness.The experiment porch applied of the design consideration of primer label is different and different, consider the feature of Illumina GA order-checking platform itself, the present invention design primer label time mainly consider following some: 1: in primer label sequence, avoid more than 3 (comprising 3) single base repetitive sequences, 2: in the same site of all primer label, the total content of base A and base C accounts between the 30%-70% of all base contentses, 3: the GC content of primer label sequence itself is between 40-60%, 4: between primer label, sequence difference degree is greater than 4 bases, 5: avoid in primer label sequence occurring the sequence high with Illumina GA sequencing primer similarity, 6: reduce primer label sequence and add to after in PCR primer, to the serious hair fastener (hairpin) that PCR primer causes, the appearance of dimer (dimer) situation.
Term " PCR-Free library joint (adapter) " refers to one section of base through design, its Main Function is that auxiliary fixed dna molecule is on sequence testing chip and provide the binding site of universal sequencing primer thing, PCR-Free library joint can be connected directly to the DNA fragmentation two ends in sequencing library by DNA ligase, the importing process of joint, because do not have the participation of PCR, is therefore called PCR-Free library joint." joint (adapter) " or " library joint (library adapter) " label technique refers to that (composition sequence of different libraries joint is different by adding different libraries joint to multiple sequencing library, sequence distinct portions is called splice tag (adapter index)), build label sequencing library, thus the mixing order-checking of multiple different label sequencing libraries can be realized, and the final a kind of library label technique that can mutually distinguish of the sequencing result in each label sequencing library.Such as, use PCR-FREE library joint from ILLUMIA in the embodiment of the present invention.
In following embodiment, by the 3 pairs of PCR primer filtered out, carry out HLA-C site pcr amplification to the blood sample of 95 routine known HLA Common genes types, amplified production checks order through sanger method and s-generation sequence measurement.Sequencing result is used for HLA-C somatotype, and by comparing the conservative property and specificity of verifying PCR primer with former genotyping result.
Embodiment 1 s-generation sequencing technologies (Illumina GA) carries out HLA-C gene type
1. sample DNA extracts
Use KingFisher automatic extracting instrument to extract in 95 routine known other blood samples of HLA genotype and extract DNA.Key step is as follows: take out the supporting deep-well plates of 6 Kingfisher automatic extracting instruments and 1 shallow bore hole plate, add a certain amount of supporting reagent respectively according to specification sheets and mark, all orifice plates having added reagent are placed in corresponding position on request, selection procedure " Bioeasy_200 μ l Blood DNA_KF.msz " program, presses " start " and performs this program and carry out nucleic acid extraction.The eluted product of collecting 100 μ about l in plate Elution after EP (end of program) is the DNA of extraction, as the template in next step PCR.
2.PCR increases
The PCR primer by synthesizing 5 ' end with different primers label makes different PCR Tag primers, and these different PCR Tag primers may be used for different samples, and described PCR primer is 2, the 3 and 4 exon PCR primer for HLA-C.Thereafter introduce primer label by PCR reaction at PCR primer two ends, thus specific marker is from the PCR primer of different sample.
To increase respectively 95 parts of DNA samples with 95 cover PCR Tag primers, often overlap PCR Tag primer to be made up of the PCR primer (table 1) of 2,3 and 4 exons for the HLA-C that increases and a pair two-way primer label (table 2), 5 ' end of wherein each forward PCR primer connects the forward primer label of pair of primers label, and 5 ' end of inverse PCR primer connects the reverse primer label of pair of primers label.Primer label is directly added on 5 ' end of PCR primer when primer synthesizes.
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 plates, numbering is respectively the site that HLA-P-C2, HLA-P-C3, HLA-P-C4(C2/3/4 represent amplification), arrange the negative control that is not added template in plate, negative control the primer is identical with primer PI-96.While experiment, record the sample number information that often pair of primer label is corresponding.
The relevant information of table 2. primer label
By in step 1 with KingFisher automatic extracting instrument extract DNA for template, have the HLA-C of label each exon primer one tube PCR amplification with 5 ' end band, PCR program is as follows:
C2:96 DEG C 2 minutes; 95 DEG C of 30s → 62 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞;
C3:96 DEG C 2 minutes; 95 DEG C 30 seconds → 56 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞;
C4:96 DEG C 2 minutes; 95 DEG C 30 seconds → 60 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞.
The PCR reaction system of HLA-C is as follows:
Promega5×buffer I(Mg2+plus) |
5.0μl |
DNTP mixture (each 2.5mM) |
2.0μl |
PI
nf-C-F
2/3/4(50ng/μl)
|
1.5μl |
PI
nf-C-R
2/3/4(50ng/μl)
|
1.5μl |
Promega Taq(5U/μl) |
0.2μl |
DNA (about 20ng/ μ l) |
2.0μl |
ddH
2O
|
12.8μl |
Amount to |
25.0μl |
Wherein PI
nf-C-F
2/3/4represent the F primer of primer 5 ' end with the HLA-C of No. n-th forward primer sequence label (table 2), PI
nf-C-R
2/3/4represent the R primer (herein n≤96) of primer 5 ' end with the HLA-C of No. n-th reverse primer sequence label, other the like.And the corresponding specific a set of PCR primer of each sample.
PCR reaction runs on the PTC-200PCR instrument of Bio-Rad company.After PCR completes, get 2 μ l PCR primer through 1.5% agarose gel electrophoresis detect.Fig. 1 shows front 20 sample HLA-C corresponding exon PCR primer electrophoresis result, DNA molecular marker is DL2000(Takara company), glue figure there is a series of clip size be the single band of 400bp-500bp, show the success of each exon of the HLA-C of this part sample (C2, C3, C4) pcr amplification.The result of other sample is similar.
The mixing of 3.PCR product and purifying
From the remaining PCR primer of 96 orifice plate HLA-P-C2, (except negative control) is respectively got 20 μ l and is blended in the EP pipe of a 3ml, be labeled as HLA-C2-Mix, same operation is carried out to other 2 96 orifice plates, be labeled as HLA-C3-Mix and HLA-C4-Mix respectively, concussion mixing, from HLA-C2-Mix, respectively getting 200 μ l in HLA-C3-Mix and HLA-C4-Mix is blended in the EP pipe of a 1.5ml, be labeled as HLA-Mix, 500 μ l DNA mixtures are got through Qiagen DNA Purification kit(QIAGEN company from HLA-Mix) cross column purification (concrete purification step refers to specification sheets), 200 μ l DNA of purifying gained, measuring HLA-Mix DNA concentration through Nanodrop8000 (Thermo Fisher Scientific company) is 50ng/ μ l.
The structure of 4.Illumina GA PCR-Free sequencing library
Interrupting of 4.1PCR product
Get from the HLA-Mix after purifying total amount 5 μ g DNA Covaris microTube with AFA fiber andSnap ?Cap interrupt at Covaris S2 (Covaris company).Interrupt condition as follows:
Frequency sweeping (frequency sweeping)
Duty ratio (Duty Cycle) |
10% |
Intensity |
3 |
Circulation/pulse (Cycles/Burst) |
200 |
Time (second) |
180 |
4.2 interrupt after PCR primer purifying
All product QIAquick PCR Purification Kit that interrupt of HLA-Mix are reclaimed purifying, are dissolved in the EB(QIAGEN Elution Buffer of 37.5 μ l respectively) in.
4.3 end reparation reactions
DNA end reparation reaction is carried out to the product of purifying, system following (reagent is all purchased from Enzymatics company):
The product of previous step purifying |
37.5μl |
10 × polynucleotide kinase damping fluid (B904) |
5μl |
DNTP mixture (often kind of 10mM) |
2μl |
T4DNA polysaccharase |
2.5μl |
Klenow fragment |
0.5μl |
T4 polynueleotide kinase |
2.5μl |
Cumulative volume |
50μl |
Reaction conditions is: at 20 DEG C, temperature bath 30 minutes in Thermomixer (Eppendorf company).
Reaction product reclaims purifying through QIAquick PCR Purification Kit, is dissolved in the EB(QIAGEN ElutionBuffer of 32 μ l) in.
4.43 ' end adds A reaction
3 ' the end that previous step reclaims DNA adds A reaction, system following (reagent is all purchased from Enzymatics company):
Previous step gained DNA |
32μl |
10 × blue damping fluid |
5μl |
DATP (1mM, GE company) |
10μl |
Klenow(3’-5’exo-) |
3μl |
Reaction conditions is: at 37 DEG C, temperature bath 30 minutes in Thermomixer.
Reaction product is through MiniElute PCR Purification Kit(QIAGEN company) reclaim purifying, be dissolved in the EB solution (QIAGEN Elution Buffer) of 38 μ l.
4.5 connect Illumina GA PCR-Free library joint (adaptor)
Add the product after A and connect Illumina GA PCR-Free library joint, system following (reagent is all purchased from Illumina company):
The DNA of previous step |
38μl |
10 × connect damping fluid |
5μl |
PCR-free oligonucleotide joint mixture (30mM) |
2μl |
T4 DNA ligase (Rapid, L603-HC-L) |
5μl |
Cumulative volume |
50μl |
Reaction conditions is: at 16 DEG C, and in Thermomixer, temperature bath is spent the night.
Reaction product is dissolved in 50 μ l deionized waters after Ampure Beads (Beckman Coulter Genomics) purifying, detects that DNA concentration results is as follows through quantitative fluorescent PCR (QPCR):
|
QPCR detected result (nM) |
HLA-Mix |
122.71 |
4.6 rubber tapping are reclaimed
Get 30 μ l HLA-Mix 2% low melting-point agarose glue to reclaim.Deposition condition is 100V, 100 minutes.DNA standard molecular weight object of reference is the 50bp DNA Ladder of NEB company.The DNA fragmentation (accompanying drawing 2) of 400-750 bp length range is reclaimed in rubber tapping.Glue reclaims product through QIAquick PCR Purification Kit(QIAGEN company) reclaim purifying, after purifying, volume is 32 μ l, detects that DNA concentration results is 17.16nM through quantitative fluorescent PCR (QPCR).
5.IlluminaGA checks order
According to QPCR detected result, get the order-checking of 10pmol DNA Illumina GAPE-100 program, concrete operations flow process refers to Illumina GA process specifications (Illumina GAIIx).
6. interpretation of result
The sequencing result of Illumina GA output is a series of DNA sequence dnas, by searching positive and negative primer label sequence in sequencing result and primer sequence, sets up the database of the corresponding sample HLA-C of each primer label each exon PCR primer sequencing result; The sequencing result of each exon is positioned at (reference sequences source: http://www.ebi.ac.uk/imgt/hla/), and build consistence (consensus) sequence of each database on the reference sequences of corresponding exon by BWA (Burrows-Wheeler Aligner); The diversity factor of combined alkali based sequencing mass value and sequencing sequence and consensus sequence, error recovery that sequencing sequence is screened and checked order; And the DNA sequence dna after correcting can be assembled into the corresponding sequence of each exon of HLA-C by overlapping sequences (overlap) and chain (Pair-End is chain) relation.The example screenshot of Fig. 3 describes the process built the 2 exon consensus sequences in the HLA-C site of No. 1 sample.
By the sequence library comparison of the DNA sequence dna of checked order HLA-C intron to the corresponding each exon of HLA-C in IMGT HLA specialized database, the HLA-C gene type being corresponding sample that sequence alignment result 100% is mated.All 95 samples, the genotyping result obtained conforms to completely with former known genotyping result, and wherein the concrete outcome of 1-32 sample and the original genotyping result of sample contrast as follows: (as shown in table 3, whole detected result is identical with original detected result).
Table 3: this genotyping result and the original genotyping result of sample contrast
Note: the C*0303 in HLA-C type does not get rid of the possibility of C*0320N, C*0401 does not get rid of the possibility of C*0409N/0430, C*0702 does not get rid of the possibility of C*0750, C*0801 does not get rid of the possibility of C*0822, C*1505 does not get rid of the possibility of C*1529 because above-mentioned allelotrope HLA-C2,3, the sequence of 4 exons is identical.
Embodiment 2: carry out HLA-C gene type with the order-checking of sanger method
1. sample DNA extracts
Similar as described in example 1 above, 26 routine known other DNA of HLA genotype in the 95 routine samples extracted with KingFisher automatic extracting instrument.
2.PCR increases
The DNA extracted with above-mentioned KingFisher automatic extracting instrument is for template, and with C-F2, C-R2, C-F3, C-R3, C-F4, C-R4 totally 3 pairs of PCR primer one tube PCR amplification respectively, each pair of primer PCR program is as follows:
C2:96 DEG C 2 minutes; 95 DEG C 30 seconds → 62 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞;
C3:96 DEG C 2 minutes; 95 DEG C 30 seconds → 56 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞;
C4:96 DEG C 2 minutes; 95 DEG C 30 seconds → 60 DEG C 30 seconds → 72 DEG C 20 seconds (35 circulations); 15 DEG C of ∞.
The PCR reaction system of HLA-C is as follows:
Promega5×buffer I(Mg2+plus) |
5.0μl |
The each 2.5mM of dNTP Mixture() |
2.0μl |
Primer mix(50ng/μl) |
3.0μl |
Promega Taq(5U/μl) |
0.2μl |
DNA (about 20ng/ μ l) |
2.0μl |
ddH
2O
|
12.8μl |
Amount to |
25.0μl |
PCR primer, after agarose gel electrophoresis detects (accompanying drawing 4), prepares purifying.
3.PCR product purification
Millipore purifying plate is utilized to carry out PCR primer purifying.Basic step is: on 96 hole PCR primer purify plate, mark the hole needing to use with marking pen, and 50 μ l ultrapure waters are added in the hole needing to use, remaining hole pastes sealed membrane, room temperature leaves standstill 15 minutes or is connected in suction filtration system,-10Pa, within 5 minutes, take off, when suction filtration system takes off purifying plate, all will blot at thieving paper the liquid remaining in leakage fluid dram bottom purifying plate at every turn.
PCR primer to be purified is centrifugal, 4000rpm, 1 minute; Open lid or the silicagel pad of PCR primer to be purified, in each PCR reaction system, add 100 μ l ultrapure waters.Then the purifying plate adding PCR primer to be purified is connected in suction filtration system, regulate vacuum tightness to weather gage display-10 handkerchiefs, absence of liquid on the micropore regenerated fibre film bottom suction filtration to purifying plate, observes under illumination, without complete liquid level specular gloss.
To need purified pcr product hole in add 50 μ l ultrapure waters or TE on micropore regenerated fibre film; To use in micro oscillator frequency modulated oscillations purifying plate under room temperature 5 minutes, in transfer respective aperture all liquid in hole corresponding to 96 new hole PCR plate.
4. carry out sequencing reaction and purifying sequencing reaction product
With the PCR primer after above-mentioned purifying for template does sequencing reaction, the condition of sequencing reaction is: 96 DEG C 2 minutes; 96 DEG C 10 seconds → 55 DEG C 5 seconds → 60 DEG C 2 minutes (25 circulations); 15 DEG C of ∞.
The system of sequencing reaction is:
PCR primer after purifying |
1μl |
Primer (3.2pmol/l) |
1μl |
2.5*Bigdye |
0.3μl |
5*Buffer |
0.85μl |
Water |
1.85μl |
Cumulative volume |
5μl |
By following steps purifying sequencing reaction product: take off the trim of sequencing reaction plate, under 3000g centrifugal 1 minute.The every 5 μ l reaction systems of 96 orifice plates add 2 μ l0.125mol/L EDTA-Na2 solution, and 33 μ l85% ethanol, cover silicagel pad, fully vibration 3 minutes, at 4 DEG C with 3000g centrifugal 30 minutes.Take out order-checking plate after centrifugal end, open silicagel pad, sequencing reaction plate is inverted on thieving paper, stops immediately when the heterogeneous example showing an absence of inverse disconnection between the middle term and the major term heart to centrifugal force reaches 185g.The 96 every holes of orifice plate add 50 μ l70% ethanol, cover silicagel pad, vibrate 1.5 minutes, at 4 DEG C with 3000g centrifugal 15 minutes.Sequencing reaction plate puts lucifuge ventilation 30 minutes, air-dry extremely without ethanol smell.The 96 every holes of orifice plate add the 10 every holes of μ l(384 orifice plate and add 8 μ l) HI-DI methane amide, cap seal membrana oralis, vibrates centrifugal to 1000rpm after 5 seconds.
5 order-checking and interpretations of result
Sequencing reaction product after purifying carries out capillary electrophoresis order-checking on ABI3730XL, and order-checking peak figure analyzes (accompanying drawing 5) through uType software (Invitrogen), obtains HLA genotyping result.Whole detected result identical with original detected result (table 4).
Table 4: this genotyping result and the original genotyping result of sample contrast
It will be apparent to one skilled in the art that and can carry out various modifications and changes to the present invention under the prerequisite not deviating from scope and spirit of the present invention.By considering the present invention's specification sheets disclosed herein and example, other embodiments of the present invention are apparent to those skilled in the art.This specification sheets and embodiment should only regard exemplary purposes as, and the real scope and spirit of the present invention illustrate in the appended claims.
Reference:
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