CN108531568B - It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method - Google Patents
It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method Download PDFInfo
- Publication number
- CN108531568B CN108531568B CN201810333623.9A CN201810333623A CN108531568B CN 108531568 B CN108531568 B CN 108531568B CN 201810333623 A CN201810333623 A CN 201810333623A CN 108531568 B CN108531568 B CN 108531568B
- Authority
- CN
- China
- Prior art keywords
- hla
- gene
- primer
- sequence
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6869—Methods for sequencing
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method, belongs to genetic test field.Provided by the present invention for the primer sets of HLA-B gene magnification, at least two groups primer is designed according to the 8 of HLA-B gene exon 1s, carries out PCR amplification using HLA-B gene described in the primer pair, obtains the product that genetic fragment length is less than 1.5kb；HLA-B gene is expanded by above-mentioned primer PCR, so that the genetic fragment length of amplified production is less than 1.5kb, the integrity demands of template are reduced, amplification efficiency is high, and the amplified reaction time is short, cost reduces, it can satisfy the amplification of extensive sample HLA-B gene or the demand of Genotyping, provide more accurate foundation for clinic HLA distribution type, provide suitable transplantation donor for patient, the rejection in migration process is reduced, the success rate and survival of organ transplant are improved.
Description
Technical field
The invention belongs to genetic test fields, and in particular to a kind of for HLA-B gene magnification, the primer of Genotyping
Group, kit and method.
Background technique
Human leukocyte antigen (human leukocyte antigen, abbreviation HLA), encoding gene are located at No. 6 chromosomes
On galianconism, overall length about 4000Kb is the genetic polymorphism system of currently known most complex human, the function of immune system with the mankind
It is closely related.HLA is otherwise known as transplantation antigen, is an important factor for determining graft-rejection height.Carrying out organ transplant
When, HLA compatibility is higher between donor and receptor, and the incidence of rejection is lower, transplants success rate and transplant organ
Patient's long-term surviving rate is higher；Conversely, being more easy to happen rejection.Therefore, the parting pair of efficiently and accurately is carried out to HLA
Organ transplant is most important.
HLA gene is divided into three classes: I class, II class and Group III gene.Wherein, I class includes: HLA-A, HLA-B, HLA-C base
Cause；II class includes: HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1 gene.
HLA classifying method includes serological typing method, cytology typing and Molecular strain typing method.With molecule
The rapid development of biology techniques, traditional serology and cytology classifying method are gradually by Molecular strain typing method institute
Replace.At this stage, HLA molecular typing methods mainly have: polymerase chain reaction,PCR-restriction fragment length polymorphism (PCR-
RFLP), polymerase chain reaction,PCR-sequence specific primers (PCR-SSP), polymerase chain reaction,PCR-sequence specific oligonucleotide are anti-
It answers (PCR-SSOP), genetic chip and sequencing and typing (PCR-SBT) etc..
HLA-B sequencing and typing (PCR-SBT) method based on polymerase chain reaction (PCR) is most accurate and reliable HLA-
1 B gene classifying method is " goldstandard " of the HLA-B methods of genotyping that the World Health Organization (WHO) recommends.
International patent documents WO2011035550A1 discloses a kind of HLA gene magnification and methods of genotyping and its related draws
Object, the patent additionally provide amplimer used in the method and the method for HLA Genotyping to and sequencing primer, answer
With amplimer provided by the invention to and sequencing primer and methods of genotyping, can be on the basis of expanding full-length gene
Genotyping is carried out, but the technology is expanded using 8 exons of the pair of primers to HLA-B gene, the overall length of HLA-B
Gene is 2.5kb, and segment is relatively long, therefore amplification difficulty is bigger, to the more demanding of DNA profiling, while needing high guarantor
Genuine enzyme, the higher cost of high fidelity enzyme are not able to satisfy the demand of extensive sample HLA-B Genotyping.
Therefore, it is necessary to one kind, cost is relatively low, the HLA-B methods of genotyping of accuracy rate and high resolution, for extensive
The HLA-B Genotyping of sample.
Summary of the invention
The technical problem to be solved in the present invention is that existing HLA-B full length gene segment is longer, DNA profiling integrality is wanted
Ask high, amplification difficulty is big, at high cost, the problem of not being able to satisfy extensive sample HLA-B parting, and the present invention provides one kind and is used for
HLA-B gene magnification, the primer sets of high-resolution parting, kit and method.
For this purpose, the present invention provides a kind of primer sets for HLA-B gene magnification, according to aobvious outside the 8 of HLA-B gene
Sub-district designs at least two groups primer, carries out PCR amplification using HLA-B gene described in the primer pair, obtains genetic fragment length
Product less than 1.5kb.
The primer sets for HLA-B gene magnification design two groups according to the 8 of HLA-B gene exon 1s and draw
Object is expanded the 1st exon of HLA-B gene to the 3rd exon using first group of primer PCR, is expanded using second group of primer PCR
Increase the 4th exon of HLA-B gene to the 8th exon.
The primer sets for HLA-B gene magnification, the nucleotide sequence of first group of primer include as follows
(a1)-(a4) any one in sequence:
(a1) sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；Or
(a2) increase for 5 ' ends of at least one sequence in sequence described in (a1) and/or 3 ' ends and be less than or equal to 8
The sequence of nucleotide；Or
(a3) it reduces for being held at 5 ' ends and/or 3 ' at least one sequence in sequence described in (a1) less than or equal to 3
The sequence of a nucleotide；Or
(a4) middle part at least one sequence in sequence described in (a1) increases, reduces and/or replaces and is less than or waits
In the sequence of 2 nucleotide；
The nucleotide sequence of second group of primer includes any one in following (b1)-(b4) sequence:
(b1) sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4；Or
(b2) increase for 5 ' ends of at least one sequence in sequence described in (b1) and/or 3 ' ends and be less than or equal to 8
The sequence of nucleotide；Or
(b3) it reduces for being held at 5 ' ends and/or 3 ' at least one sequence in sequence described in (b1) less than or equal to 3
The sequence of a nucleotide；Or
(b4) increase, reduce and/or replace the sequence for being less than or equal to 2 nucleotide for the middle part of sequence described in (b1)
Column.
Preferably, the primer sets for HLA-B gene magnification, above-mentioned increase, reduction and/or substituted nucleotide
Selected from A, T, C or G.
The primer sets for HLA-B gene magnification, first group of primer PCR amplification HLA-B genetic fragment are long
Degree is 1.2kb, and second group of primer PCR amplification HLA-B genetic fragment length is 1.4kb.
The present invention provides a kind of kits for HLA-B gene magnification, are used for HLA-B gene magnification including described
Primer sets.
The kit for HLA-B gene magnification, the PCR reaction system including HLA-B gene magnification, with 20 μ L
It is as follows for meter:
2 × PCR buffer, 10 μ l；
Primer B-F1,5 μM, 0.8 μ l；
Primer B-R1,5 μM, 0.8 μ l；
Primer B-F2,5 μM, 0.8 μ l；
Primer B-R2,5 μM, 0.8 μ l；
DNA profiling, 20-50ng/ μ l, 2 μ l；
Taq enzyme, 5U/ μ L, 0.15 μ l；
Surplus complements to 20 μ L with pure water.
Preferably, the kit for HLA-B gene magnification, the PCR reaction system of the HLA-B gene magnification
In, primer Invitrogen 10X PCR Buffer (- MgCl2) 100 times of diluteds to 5 μM.
Preferably, the kit for HLA-B gene magnification, the PCR reaction system of the HLA-B gene magnification
In, 2 × PCR buffer formula is as follows, by 10 μ l in terms of: dNTP, 25mM, 0.16 μ l；MgCl2, 20-30mM, 1.8 μ l；DTT,
1M, 0.03 μ l；100%DMSO, 0.27 μ l；Betain, 5M, 2.16 μ l；BSA, 20mg/ml, 0.01 μ l；Thermo 10X Taq
Bufferwith(NH4)2SO4, 2 μ l；Surplus complements to 10 μ L with pure water.
The present invention provides a kind of method of HLA-B gene magnification, including the use of described for HLA-B gene magnification
Primer sets or the kit PCR amplification HLA-B gene for HLA-B gene magnification.
The PCR response procedures of the method for the HLA-B gene magnification, the HLA-B gene magnification are as follows: 96 DEG C of pre- changes
Property 2min；96 DEG C of holding 30Sec (second), 65 DEG C of holding 30Sec (second), 72 DEG C of holding 2min react 5 circulations；96 DEG C of holdings
30Sec (second), 62 DEG C of holding 30Sec (second), 72 DEG C of holding 2min react 35 circulations；10 DEG C of preservations.
The present invention provides a kind of methods of HLA-B High Resolution Gene Typing, include the following steps:
S1, sample to be tested DNA is obtained from people in vitro tissue or blood；
S2, the primer sets for HLA-B gene magnification, the reagent for HLA-B gene magnification are utilized
Box and/or the method PCR amplification HLA-B gene of the HLA-B gene magnification；
S3, the PCR product amplified in S2 step is purified and is sequenced；
S4, the sequencing result in step S3 is compared with standard HLA-B gene order, determines the HLA- of sample to be tested
The type of 1 B gene.
The method of the HLA-B High Resolution Gene Typing is sequenced using Sanger method in the S3 step.
The method of the HLA-B High Resolution Gene Typing, the nucleosides of the Sanger sequencing primer used in the S3 step
Acid sequence includes any one in following sequence:
(c1) sequence is as shown in SEQ ID NO.5-SEQ ID NO.10；Or
(c2) increase for the 5 ' ends, 3 ' ends of at least one sequence in sequence described in (c1) and/or middle part, reduce and/
Or replace the sequence of 1 nucleotide.
Amplification reaction system is sequenced in the method for the HLA-B High Resolution Gene Typing, in the S3 step, Sanger, with
10 μ L are to be calculated as:
Sequencing primer, 5 μM, 0.75 μ l；
Sequencing template DNA, 2.0 μ l；
BigDye Terminator v3.1Ready Reaction Mix (ABI), 0.25 μ l；
Sequencing buffer, 3.0 μ l；
Add ddH2O to 10 μ l.
Preferably, the method for the HLA-B High Resolution Gene Typing, the Sanger sequencing primer Invitrogen
10X PCR Buffer(-MgCl2) 100 times of diluteds to 5 μM.
Preferably, the method for the HLA-B High Resolution Gene Typing, the Sequencing buffer formula are as follows:
5X sequencing Buffer (ABI), 2 μ l；Betain, 5M, 1 μ l；DNTPs, 20-25mM, 0.0005 μ l.
The method of the HLA-B High Resolution Gene Typing, in the S3 step, it is as follows that amplification program is sequenced in Sanger:
96 DEG C of initial denaturation 1min；96 DEG C of holdings 10Sec, 60 DEG C of holding 2min react 40 circulations；10 DEG C of preservations.
Preferably, the method for the HLA-B High Resolution Gene Typing, in the S3 step, Sanger sequencing include as follows
Step:
Q1, the amplified production for obtaining step S2 carry out digestion purifying, and used enzyme is ThermoFisher FastAP
Thermolabile alkaline phosphatase and NEB Exonuclease I (E.coli)；
Q2, using the sequencing primer, the purified product that step Q1 is obtained carries out PCR reaction；
Then Q3, the amplified production for obtaining step Q2 carry out Capillary Electrophoresis order-checking using ethyl alcohol/EDTA method purifying.
The present invention provides a kind of sequencing primer groups for HLA-B High Resolution Gene Typing, including in following sequence
Any one:
(c1) sequence is as shown in SEQ ID NO.5-SEQ ID NO.10；Or
(c2) increase for the 5 ' ends, 3 ' ends of at least one sequence in sequence described in (c1) and/or middle part, reduce and/
Or replace the sequence of the sequence of 1 nucleotide.Wherein, sequencing primer SEQ IDNO.5-SEQ ID NO.6 is for being sequenced HLA-B
2 exons of gene, sequencing primer SEQ IDNO.7-SEQ ID NO.8 are used to be sequenced 3 exons of HLA-B gene, survey
Sequence primer SEQ IDNO.9-SEQ ID NO.10 is used to be sequenced 4 exons of HLA-B gene.
Preferably, the primer sets for HLA-B gene magnification, above-mentioned increase, reduction and/or substituted nucleotide
Selected from A, T, C or G.
The present invention provides a kind of kits for HLA-B High Resolution Gene Typing, are used for HLA-B base including described
The primer sets of gene-amplification, the described kit for HLA-B gene magnification and/or described it is used for HLA-B High Resolution Gene
The sequencing primer group of parting.
The kit for HLA-B High Resolution Gene Typing further includes sequencing amplification reaction system, is with 10 μ L
It is calculated as:
Sequencing primer, 5 μM, 0.75 μ l；
Sequencing template DNA, 2.0 μ l；
BigDye Terminator v3.1Ready Reaction Mix (ABI), 0.25 μ l；
Sequencing buffer, 3.0 μ l；
Add ddH2O to 10 μ l.
Technical solution of the present invention has the advantages that
1, it provided by the present invention for the primer sets of HLA-B gene magnification, is set according to the 8 of HLA-B gene exon 1s
At least two groups primer is counted, PCR amplification is carried out using HLA-B gene described in the primer pair, obtains genetic fragment length and be less than
The product of 1.5kb；HLA-B gene is expanded by above-mentioned primer PCR, so that the genetic fragment length of amplified production is less than 1.5kb,
The integrity demands of template are reduced, amplification efficiency is high, and the amplified reaction time is short, and cost reduces, and can satisfy extensive sample
The amplification of HLA-B gene or the demand of Genotyping.
2, it provided by the present invention for the primer sets of HLA-B gene magnification, is set according to the 8 of HLA-B gene exon 1s
Two groups of primers are counted, using first group of primer PCR amplification the 1st exon of HLA-B gene to the 3rd exon, using second group
Primer PCR expands the 4th exon of HLA-B gene to the 8th exon；Using above-mentioned primer sets respectively to HLA-B gene
Exons 1-3 and exon 4-8 carry out specific amplification, ensure that the mrna length of two segments of amplification is close, avoid base
Because length difference is away from excessive, cause amplification system unstable, increases amplification difficulty, further improve amplification efficiency, shorten amplification
Reaction time.
3, provided by the present invention for the primer sets of HLA-B gene magnification, the nucleotide sequence packet of first group of primer
Include any one in following (a1)-(a4) sequence: (a1) sequence is as shown in SEQ IDNO.1 and SEQ ID NO.2；Or (a2)
Increase the sequence for being less than or equal to 8 nucleotide for 5 ' ends of at least one sequence in sequence described in (a1) and/or 3 ' ends；
Or (a3) is that holding at 5 ' ends and/or 3 ' at least one sequence is reduced less than or equal to 3 nucleosides in sequence described in (a1)
The sequence of acid；Or (a4) be at least one sequence in sequence described in (a1) middle part increase, reduce and/or replace be less than or
Equal to the sequence of 2 nucleotide；The nucleotide sequence of second group of primer includes any one in following (b1)-(b4) sequence
Kind: (b1) sequence is as shown in SEQ ID NO.1 and SEQID NO.2；Or (b2) is at least one sequence in sequence described in (b1)
5 ' the ends and/or 3 ' ends of column increase the sequence for being less than or equal to 8 nucleotide；Or (b3) be described in (b1) in sequence at least
One holding at 5 ' ends and/or 3 ' for sequence reduces the sequence for being less than or equal to 3 nucleotide；Or (b4) is sequence described in (b1)
The middle part of at least one sequence increases, reduces and/or replaces the sequence for being less than or equal to 2 nucleotide in column；In order to expand
The full exon for increasing HLA-B gene out guarantees that the allele of all HLA-B genes is all amplified, and improves amplification efficiency, simple
Change amplification system, reduce amplification cost, above-mentioned primer is designed according to following thinkings:
(1) to guarantee that the allele of all HLA-B is all amplified, need to choose conserved region sequence design primer, simultaneously
HLA gene polynorphisms are also contemplated, primer is set to the conserved region without mutational site as far as possible, if can not find suitable conservative
Area then uses and annexs base or add primer to guarantee effective amplification of all allele, avoids missing inspection genotype.
(2) amplification efficiency maximizes, and controls the base composition of primer first, avoids repetitive sequence, and control G/C content exists
Within the scope of 40%-60%, and primer both ends base avoids the occurrence of A/T.Meanwhile the solution temperature between the primer in same system
Difference should not be too large.Secondly, control amplified production length, amplified production length are less than 1500bp.
(3) amplification system is most simplified, to reduce the number of target fragment in same amplification system, if amplified fragments mistake
More, it is excessive to will cause system inner primer item number, will increase the possibility interfered with each other between primer, and also add test operation
Complexity.
(4) cost minimization is expanded, needs to realize by two aspects, improves the amplification efficiency of primer first, it in this way can be with
It avoids using expensive special archaeal dna polymerase.In addition, also to control the length of amplified production, if length is too long, need
Specific enzyme is expanded using the more expensive long segment of price, to increase cost.
(5) by analyzing HLA-B gene order, in summary factor, determines that best amplification scheme is as follows: will
The amplification of HLA-B gene is divided to two segments to carry out, the two segments are exons 1-3 (1200bp) and exon 4-8 respectively
(1400bp).The reason is as follows that: firstly, the amplification of HLA-B gene is divided into above-mentioned two segment, it is not only long in reduction amplified fragments
The requirement for reducing amplified fragments is had also contemplated while spending.If not being segmented amplification, amplified fragments are too long (> 3000bp), make
It is larger at amplification difficulty.Such as it is divided into exons 1-2 and exon 3-8 if be segmented otherwise, causes one of piece
Section is too long, also increases amplification difficulty.If being divided into 3 sections or more amplifications, need to add more primers, increases between primer
Effect is interfered with each other, to reduce amplification efficiency.Secondly, there are conserved region in the 5 ' NCR, introne 3 and 3 ' NCR of HLA-B,
The conserved region sequence meets above-mentioned primer sequence requirement.Therefore, pass through comprehensive and balance various factors, it is determined that above-mentioned draws
Object sequence.
4, a kind of kit for HLA-B gene magnification provided by the invention expands including described for HLA-B gene
The primer sets of increasing carry out PCR amplification to the HLA-B gene using the primer sets, obtain genetic fragment length less than 1.5kb
Product；HLA-B gene is expanded by above-mentioned primer PCR, so that the genetic fragment length of amplified production is less than 1.5kb, to mould
The integrity demands of plate reduce, and amplification efficiency is high, and the amplified reaction time is short, and cost reduces, and can satisfy extensive sample HLA-B
The amplification of gene or the demand of Genotyping.
5, a kind of kit for HLA-B gene magnification provided by the invention, the PCR including HLA-B gene magnification are anti-
System is answered, it is as follows: 2 × PCR buffer, 10 μ l by 20 μ l in terms of；Primer B-F1,5 μM, 0.8 μ l；Primer B-R1,5 μM, 0.8
μl；Primer B-F2,5 μM, 0.8 μ l；Primer B-R2,5 μM, 0.8 μ l；Taq enzyme, 5U/ μ L, 0.15 μ l；Surplus is complemented to pure water
20μl；It does not need to expand dedicated valuableness, high heat stability and hi-fi using long segment in above-mentioned PCR reaction system
Archaeal dna polymerase, and two segment while property amplifications can be completed in a reaction system, therefore do not increasing operating procedure
On the basis of, significantly reduce cost.
6, the method for HLA-B gene magnification provided by the invention, including the use of the drawing for HLA-B gene magnification
Object group or the kit PCR amplification HLA-B gene for HLA-B gene magnification, the product of amplification is shorter (< 1.5kb),
The integrity demands of template are reduced, amplification efficiency is high, and the amplified reaction time is short.
7, the method for HLA-B High Resolution Gene Typing provided by the invention includes the following steps: S1, from the in vitro group of people
Knit or blood in obtain sample to be tested DNA；S2, using the described primer sets for HLA-B gene magnification, described be used for
The kit of HLA-B gene magnification and/or the method PCR amplification HLA-B gene of the HLA-B gene magnification；S3, by step
The PCR product amplified in S2 is purified and is sequenced；S4, by the sequencing result and standard HLA-B gene order in step S3
It is compared, determines the type of the HLA-B gene of sample to be tested；Pass through the primer sets PCR amplification HLA-B base in the above method
Cause reduces the integrity demands of template so that the genetic fragment length of amplified production is less than 1.5kb, and amplification efficiency is high, amplification
Reaction time is short, and cost reduces, and can satisfy the amplification of extensive sample HLA-B gene or the demand of Genotyping, for clinic
HLA distribution type provides more accurate foundation, provides suitable transplantation donor for patient, reduces the rejection in migration process,
Improve the success rate and survival of organ transplant.
8, the kit provided by the present invention for HLA-B High Resolution Gene Typing includes described for HLA-B base
The primer sets of gene-amplification, the described kit for HLA-B gene magnification and/or described it is used for HLA-B High Resolution Gene
The sequencing primer group of parting can match with most of Taq enzymes on the market, not only effectively reduce cost, increase simultaneously
Kit applicability.
9, the kit provided by the present invention for HLA-B High Resolution Gene Typing can satisfy extensive HLA-B gene
The demand of parting has many advantages, such as efficient, quick.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is primer sets amplification region schematic diagram in the embodiment of the present invention 1；
Wherein E, Exon exon；In, intron introne；F, fragment, amplified fragments；
Fig. 2 is 8 sample HLA-B gene 1-8 exon multiple PCR products electrophoresis detection knots in the embodiment of the present invention 1
Fruit；
Fig. 3 is the result that in the embodiment of the present invention 8 96 samples are carried out with HLA-B High Resolution Gene Typing；
Description of symbols:
E- exon；In- introne；F- amplified fragments.
Specific embodiment
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.Primer involved in following embodiments is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
The amplification of 1 HLA-B gene of embodiment
1. sample DNA extracts
The other blood sample of HLA-B genotype known to 96 parts is extracted using QlAamp blood extracts kit (QIAGEN)
DNA.Concentration mensuration is carried out to the DNA sample of extraction using ultraviolet specrophotometer, by extraction obtain DNA sample concentration adjust to
20-50ng/μl。
2. designing HLA-B gene magnification primer
According to HLA-B gene sequence newest in IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla/)
Column find multiple groups (every group two) suitable conservative region as shown in Figure 1, HLA-B gene has 8 exons, and guarantee every
Group conservative region can cover 8 exon 1s of HLA-B gene.In the multiple groups conservative region found, it is suitable to separately design
Amplimer, when encounter several allelic sequences it is inconsistent when, using degeneracy base, or one new allele of design
Group-specific amplification primer.Most of all, guaranteeing that the every group of candidate drugs designed have similar physical characteristic and reaction
Dynamics, to be carried out amplification reaction under identical conditions.Two groups of primers are determined by screening, and first group of amplimer is B-
F1 and B-R1 (there are S, S are to indicate C or G in B-R1 sequence, core sequence is respectively such as SEQ ID NO.1 and SEQ ID NO.2 institute
Show, wherein S present in SEQ ID NO.2 (primer B-R1) sequence is to indicate nucleotide C or G, utilizes first group of amplimer
Exons 1-3, the second group amplimer that HLA-B gene can be amplified is B-F2 and B-R2, and core sequence is respectively such as SEQ
Shown in ID NO.3 and SEQ ID NO.4, the exon 4-8 of HLA-B gene can be amplified using second group of amplimer.
The amplification of 3.HLA-B gene PCR
(1) PCR reaction mixture shown in table 1 is added to the centrifuge tube marked.First by 2 × PCRbuffer, B-
F1, B-R1, B-F2 and B-R2 are configured to PCR amplification premixed liquid, then by the DNA sample diluted, PCR amplification premixed liquid and
Taq enzyme is made into PCR reaction solution.Primer Invitrogen 10X PCR Buffer (- MgCl in table 12) 100 times dilution
Liquid is diluted to 5 μM.It wherein, include the B-R1 primer of 2 kinds of equivalent in the dilution of primer B-R1, i.e., the first is sequence SEQID
S in NO.2 is base C；It is for second S in sequence SEQ ID NO.2 is bases G.
Table 1:PCR amplification reaction system
| Reagent name | Volume |
| 2×PCR buffer | 10μl |
| B-F1(5μM) | 0.8μl |
| B-R1(5μM) | 0.8μl |
| B-F2(5μM) | 0.8μl |
| B-R2(5μM) | 0.8μl |
| DNA profiling (20-50ng/ μ l) | 2μl |
| Taq enzyme (5U/ μ L) | 0.15μl |
| ddH2O | 4.65μl |
| Total volume | 20μL |
2 × PCR buffer formula such as the following table 2 in above-mentioned table 1,
Table 2:2 × PCR buffer formula
(2) PCR reaction solution in step (1) is put into progress PCR reaction on 9700 instrument of GeneAmp PCR system,
Amplification condition is as follows:
(3) agarose gel electrophoresis of 5 μ l progress 1.6% is taken to detect the PCR reaction product that step (2) obtains.Fig. 2 is aobvious
Shown the HLA-B gene PCR amplified band of wherein 8 samples: each amplification sample has two specific amplification bands, under
Square band length is 1.2kb, indicates the length of the 1st exon to the 3rd exon；Top band length is 1.4kb, indicates the 4th
Exon to the 8th exon length.The result of other samples is identical with this.
The above results show that two groups of amplimers the exons 1-3 to HLA-B gene and exon 4-8 can carry out respectively
Specific amplification, while two reactions can carry out in the same reaction system, therefore substantially reduce proliferation time, simplify
Reaction step significantly reduces cost.
The amplification of 2 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that the 5 ' ends that first group of amplimer is B-F1 and B-R1
And/or 3 ' end increase be less than or equal to 8 nucleotide sequence, in the present embodiment for respectively B-F1 and B-R1 5 ' end and
3 ' end 8 nucleotide of each increase, the nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G, and (bird is fast
Purine), 5 ' → the 3 ' of 5 ' ends increase GGCTACAT in the present embodiment, and 5 ' → the 3 ' of 3 ' ends increase TTTGAACC, utilize first group
Amplimer can amplify HLA-B gene exons 1-3, the second group amplimer be B-F2 and B-R2 5 ' end and/or
3 ' ends increase the sequence for being less than or equal to 8 nucleotide, in the present embodiment for respectively at the 5 ' ends of B-F2 and B-R2 and 3 ' ends
Increase the sequence of 1 nucleotide, 5 ' → the 3 ' of 5 ' ends increase G in the present embodiment, and 5 ' → the 3 ' of 3 ' ends increase C, utilize second
Group amplimer can amplify the exon 4-8 of HLA-B gene.The result and implementation of gene magnification are carried out using above-mentioned primer
Amplification in example 1 is consistent.
The amplification of 3 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that the 5 ' ends that first group of amplimer is B-F1 and B-R1
And/or 3 ' end increase be less than or equal to 8 nucleotide sequence, in the present embodiment for respectively B-F1 and B-R1 5 ' end and
3 ' ends increase by 1 nucleotide, and the nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine),
5 ' ends increase C in the present embodiment, and 3 ' ends are increased G, the exon of HLA-B gene can be amplified using first group of amplimer
1-3, the 5 ' ends and/or 3 ' ends that second group of amplimer is B-F2 and B-R2 increase the sequence for being less than or equal to 8 nucleotide,
It is held in the present embodiment for 5 ' respectively in B-F2 and B-R2 and 3 ' holds the sequence for increasing by 8 nucleotide, in the present embodiment 5 ' end
5 ' → 3 ' increase CACAGTGT, 3 ' end 5 ' → 3 ' increase CCCAGAAG, can be amplified using second group of amplimer
The exon 4-8 of HLA-B gene.The result for carrying out gene magnification using above-mentioned primer is consistent with the amplification in embodiment 1.
The amplification of 4 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that the 5 ' ends that first group of amplimer is B-F1 and B-R1
And/or 3 ' end reduce be less than or equal to 3 nucleotide sequence, in the present embodiment for respectively B-F1 and B-R1 5 ' end and
3 nucleotide are reduced by 3 ' ends, and exons 1-3, the second group amplification of HLA-B gene can be amplified using first group of amplimer
The sequence for being less than or equal to 3 nucleotide is reduced at the 5 ' ends and 3 ' ends that primer is B-F2 and B-R2, in the present embodiment for respectively
The sequence of 1 nucleotide is reduced at the 5 ' ends of B-F2 and B-R2 and 3 ' ends, HLA-B can be amplified using second group of amplimer
The exon 4-8 of gene.The result for carrying out gene magnification using above-mentioned primer is consistent with the amplification in embodiment 1.
The amplification of 5 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that first group of amplimer is the middle part of B-F1 and B-R1
Increase, reduce and/or replace the sequence for being less than or equal to 2 nucleotide, in the present embodiment for respectively B-F1's and B-R1
Middle part, which increases nucleotide described in 2 nucleotide, can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine),
It is B-F2's and B-R2 using exons 1-3, the second group amplimer that first group of amplimer can amplify HLA-B gene
Middle part reduces and/or replaces the sequence for being less than or equal to 2 nucleotide, in the present embodiment for respectively in B-F2 and B-R2
Portion reduces the sequence of 1 nucleotide, and the exon 4-8 of HLA-B gene can be amplified using second group of amplimer.Using upper
The result for stating primer progress gene magnification is consistent with the amplification in embodiment 1.
The amplification of 6 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that first group of amplimer is the middle part of B-F1 and B-R1
Increase, reduce and/or replace the sequence for being less than or equal to 2 nucleotide, in the present embodiment for respectively B-F1's and B-R1
1 nucleotide is reduced by middle part, and exons 1-3, the second group amplification of HLA-B gene can be amplified using first group of amplimer
Primer be B-F2 and B-R2 middle part reduce and/or replace be less than or equal to 2 nucleotide sequence, in the present embodiment for point
The sequence for increasing by 1 nucleotide at the middle part of B-F2 and B-R2, increases T in the middle part of two primers in the present embodiment, utilizes the
Two groups of amplimers can amplify the exon 4-8 of HLA-B gene.The result and reality of gene magnification are carried out using above-mentioned primer
The amplification applied in example 1 is consistent.
The amplification of 7 HLA-B gene of embodiment
The present embodiment is substantially the same manner as Example 1, and difference is only that first group of amplimer is the middle part of B-F1 and B-R1
Increase, reduce and/or replace the sequence for being less than or equal to 2 nucleotide, in the present embodiment for respectively B-F1's and B-R1
Middle part replaces 1 nucleotide, and exons 1-3, the second group amplification of HLA-B gene can be amplified using first group of amplimer
Primer be B-F2 and B-R2 middle part reduce and/or replace be less than or equal to 2 nucleotide sequence, in the present embodiment for point
The sequence for not replacing 2 nucleotide at the middle part of B-F2 and B-R2, can amplify HLA-B gene using second group of amplimer
Exon 4-8.The result for carrying out gene magnification using above-mentioned primer is consistent with the amplification in embodiment 1.
Embodiment 8 is based on the HLA-B Genotyping an of generation (Sanger method) sequencing technologies
(1) the pure acidifying and dilution of PCR product
1.6 μ l Thermo Fisher FastAP and 0.4 μ l NEB Exonuclease I (E.coli) are taken to be added respectively
In obtained each pcr amplification product into embodiment 1, sample panel is put into PCR instrument, starts enzyme purification program: 37 DEG C of temperature
Educate 15min, 85 DEG C of enzyme-deactivating 15min.After the completion of purifying procedure, sample is diluted according to 1:3 ratio with sterile water.
(2) sequencing amplification
The sequencing amplification reaction system of HLA-B gene extron 2,3 and 4 is prepared respectively, and reaction system is as shown in table 3.
Table 3: sequencing amplification reaction system
The sequencing forward primer of HLA-B gene extron 2,3 and 4 is respectively SB-2F, SB-3F and SB-4F, core sequence
Respectively as shown in SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9；The sequencing reverse primer of exon 2,3 and 4 is distinguished
For SB-2R, SB-3R and SB-4R, core sequence is respectively as shown in SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10.
Wherein the S in SB-3R primer (SEQ ID NO.8) is to indicate nucleotide C or G, the primer dilution be include 2 kinds of equivalent SB-
S in 3R primer, i.e. SB-3R primer (SEQ ID NO.8) indicates the S in nucleotide C and SB-3R primer (SEQ ID NO.8)
Indicate two kinds of primers of nucleotide G.Above-mentioned sequencing primer Invitrogen 10X PCR Buffer (- MgCl2) 100 times
Diluted is to 5 μM.Sequencing buffer formula in above-mentioned table 2 is as follows: 5X sequencing Buffer
(ABI)
2μl、5M Betain 1μl、20-25mM dNTPs 0.0005μl。
By sequencing primer, Sequencing buffer and ddH2O is pre-configured to sequencing premixed liquid, later by 7.75 μ l
It is sequenced premixed liquid (table 3), 0.25 μ l BigDyeTM Terminator Mix(CycleSequencing Kit Version
3.1), the diluted PCR product mixing of purifying in 2 μ l steps (1), after concussion mixes, sample is placed on GeneAmp by brief centrifugation
On PCR system 9700 (AppliedBiosystems) instrument, sequencing amplification is carried out according to following sequencing amplification program:
(3) sequencing amplified production purifying
Sequencing amplified production obtained in step (2) is purified using ethyl alcohol/EDTA method, the specific steps are as follows:
A. the 125mM EDTA of 2.5 μ l is added into sequencing amplified production, concussion mixes, and maximum (top) speed is centrifuged 15s；
B. 30 μ l dehydrated alcohols are added, mix well, room temperature avoid light place 10min；
C. at 4 DEG C, 2250g is centrifuged 30min；
D. reaction tube is buckled to, revolving speed 100g is centrifuged 1min；
E. 80% ethyl alcohol of 50 μ l Fresh is added into reaction tube, later at 4 DEG C, 2250g is centrifuged 30min；
F. reaction tube is buckled to, 100g is centrifuged 1min；
G. reaction tube, room temperature avoid light place 10min, until ethyl alcohol thoroughly volatilizees are taken out；
H. the 10 high-purity formamides of μ l are added into reaction tube, after mixing fullys shake, brief centrifugation, machine sequencing in preparation.
(4) machine is sequenced on
Purified sample in step (3) is put into ABI 3130xl sequenator and is sequenced；
As further denaturation, purified sample in step (3) can also be put into ABI 3730xl sequenator
It is sequenced.
Embodiment 9 is based on the HLA-B Genotyping an of generation (Sanger method) sequencing technologies
The present embodiment is substantially the same manner as Example 8, and difference is only that described sequencing primer SB-2F, SB-3F, SB-4F, SB-
5 ' ends, 3 ' ends and/or the middle part increase of 2R, SB-3R and SB-4R, the sequence for reducing and/or replacing 1 nucleotide, in this implementation
5 ' the ends of sequencing primer SB-2F, SB-3F, SB-4F, SB-2R, SB-3R and SB-4R described in example increase by 1 nucleotide, described
Nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine), select A in the present embodiment.Using
The result that above-mentioned primer carries out gene sequencing is consistent with the sequencing result of embodiment 8.
Embodiment 10 is based on the HLA-B Genotyping an of generation (Sanger method) sequencing technologies
The present embodiment is substantially the same manner as Example 8, and difference is only that described sequencing primer SB-2F, SB-3F, SB-4F, SB-
5 ' ends, 3 ' ends and/or the middle part increase of 2R, SB-3R and SB-4R, the sequence for reducing and/or replacing 1 nucleotide, in this implementation
3 ' the ends of sequencing primer SB-2F, SB-3F, SB-4F, SB-2R, SB-3R and SB-4R described in example increase by 1 nucleotide, described
Nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine), select T in the present embodiment.Using
The result that above-mentioned primer carries out gene sequencing is consistent with the sequencing result of embodiment 8.
Embodiment 11 is based on the HLA-B Genotyping an of generation (Sanger method) sequencing technologies
The present embodiment is substantially the same manner as Example 8, and difference is only that described sequencing primer SB-2F, SB-3F, SB-4F, SB-
5 ' ends, 3 ' ends and/or the middle part increase of 2R, SB-3R and SB-4R, the sequence for reducing and/or replacing 1 nucleotide, in this implementation
5 ' the ends of sequencing primer SB-2F, SB-3F, SB-4F, SB-2R, SB-3R and SB-4R described in example and 3 ' end 1 nucleosides of each increase
Acid, the nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine), and in the present embodiment 5 '
End selection C, 3 ' end selection G.The result for carrying out gene sequencing using above-mentioned primer is consistent with the sequencing result of embodiment 8.
Embodiment 12 is based on the HLA-B Genotyping an of generation (Sanger method) sequencing technologies
The present embodiment is substantially the same manner as Example 8, and difference is only that described sequencing primer SB-2F, SB-3F, SB-4F, SB-
5 ' ends, 3 ' ends and/or the middle part increase of 2R, SB-3R and SB-4R, the sequence for reducing and/or replacing 1 nucleotide, in this implementation
The middle part of sequencing primer SB-2F, SB-3F, SB-4F, SB-2R, SB-3R and SB-4R described in example increase by 1 nucleotide, described
Nucleotide can be selected from A (adenine), T (thymidine), C (cytimidine) or G (guanine), select G in the present embodiment.Using
The result that above-mentioned primer carries out gene sequencing is consistent with the sequencing result of embodiment 8.
13 interpretation of result of embodiment
With reference to the newest HLA database of IMGT, using professional parting software JSI SeqPilot to the sequencing result of example 8
It analyses and compares, obtains the genotyping result of HLA-B gene, as shown in Figure 3.The genotyping result of all 96 samples of this implementation
High-resolution genotyping result standard is reached, and completely the same with known sample HLA-B genotyping result.
Comparative example 1
It is carried out using methods of genotyping (being denoted as X1) disclosed in international patent documents WO2011035550A1 embodiment 1
Genotyping.
With the sample of known parting, compared using the method for the present invention and X1 classifying method, it is consistent with known genotyping result.
PCR amplification: using X1 method, and gene magnification difficulty is big, and must use high-fidelity Taq enzyme, and the method for the present invention, it is only necessary to
Purpose band can be amplified with general T aq enzyme.The method of the present invention expanding effect is good, as shown in Fig. 2, can be in electrophoretogram
Clearly seeing pcr amplification product is 1.2kb (exons 1-3) and two specific bands of 1.4kb (exon 4-8).
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Sequence table
<110>sea Beijing Nuo Shikang Genentech, Inc.
<120>a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method
<130> HA201800466
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial synthesized (B-F1)
<400> 1
ggtcccagtt ctaaagtccc cacg 24
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized (B-R1)
<400> 2
tccattcaas ggagggcgac 20
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized (B-F2)
<400> 3
tgttccccgc tcagagactc 20
<210> 4
<211> 21
<212> DNA
<213>artificial synthesized (B-R2)
<400> 4
cacacgcgaa acatcccaat c 21
<210> 5
<211> 17
<212> DNA
<213>artificial synthesized (SB-2F)
<400> 5
gcgccgggag gagggtc 17
<210> 6
<211> 19
<212> DNA
<213>artificial synthesized (SB-2R)
<400> 6
ggatggggag tcgtgacct 19
<210> 7
<211> 17
<212> DNA
<213>artificial synthesized (SB-3F)
<400> 7
acggggctga ccgcggg 17
<210> 8
<211> 20
<212> DNA
<213>artificial synthesized (SB-3R)
<400> 8
aasggagggc gacattctag 20
<210> 9
<211> 20
<212> DNA
<213>artificial synthesized (SB-4F)
<400> 9
ggtcacatgg gtggtcctag 20
<210> 10
<211> 20
<212> DNA
<213>artificial synthesized (SB-4R)
<400> 10
ggctcctgct ttccctgaga 20
Claims (13)
1. a kind of primer sets for HLA-B gene magnification, which is characterized in that designed according to the 8 of HLA-B gene exon 1s
Two groups of primers carry out PCR amplification using HLA-B gene described in the primer pair, obtain the production that genetic fragment length is less than 1.5kb
Object；The 1st exon of HLA-B gene is expanded to the 3rd exon using first group of primer PCR, is expanded using second group of primer PCR
Increase the 4th exon of HLA-B gene to the 8th exon；The nucleotide sequence of first group of primer includes following (a1) institute
Show:
(a1) sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；
The nucleotide sequence of second group of primer includes shown in following (b1):
(b1) sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
2. the primer sets according to claim 1 for HLA-B gene magnification, which is characterized in that first group of primer
PCR amplification HLA-B genetic fragment length is 1.2kb, and second group of primer PCR amplification HLA-B genetic fragment length is
1.4kb。
3. a kind of kit for HLA-B gene magnification, which is characterized in that be used for HLA- including of any of claims 1 or 2
The primer sets of 1 B gene amplification.
4. the kit according to claim 3 for HLA-B gene magnification, which is characterized in that expand including HLA-B gene
The PCR reaction system of increasing is as follows by 20 μ l in terms of:
2 × PCR buffer, 10 μ l；
Primer B-F1,5 μM, 0.8 μ l；
Primer B-R1,5 μM, 0.8 μ l；
Primer B-F2,5 μM, 0.8 μ l；
Primer B-R2,5 μM, 0.8 μ l；
DNA profiling, 20-50ng/ μ l, 2 μ l；
Taq enzyme, 5 U/ μ L, 0.15 μ l；
Surplus complements to 20 μ l with pure water.
5. a kind of method of HLA-B gene magnification, which is characterized in that be used for HLA-B including the use of of any of claims 1 or 2
The kit PCR amplification HLA-B base of HLA-B gene magnification is used for described in the primer sets or claim 3 or 4 of gene magnification
Cause.
6. the method for HLA-B gene magnification according to claim 5, which is characterized in that the HLA-B gene magnification
PCR response procedures are as follows: 96 DEG C of initial denaturation 2min；96 DEG C of holdings 30Sec, 65 DEG C of holdings 30 Sec, 72 DEG C of holding 2min, reaction
5 circulations；96 DEG C of holdings 30 Sec, 62 DEG C of holdings 30 Sec, 72 DEG C of holding 2min react 35 circulations；10 DEG C of preservations.
7. a kind of method of HLA-B High Resolution Gene Typing, which comprises the steps of:
S1, sample to be tested DNA is obtained from people in vitro tissue or blood；
S2, using being used to be used for described in the primer sets of HLA-B gene magnification, claim 3 or 4 described in as claimed in claim 1 or 22
The kit of HLA-B gene magnification and/or the method PCR amplification HLA-B base of HLA-B gene magnification described in claim 5 or 6
Cause；
S3, the PCR product amplified in step S2 is sequenced；
S4, the sequencing result in step S3 is compared with standard HLA-B gene order, determines the HLA-B base of sample to be tested
The type of cause.
8. the method for HLA-B High Resolution Gene Typing according to claim 7, which is characterized in that in the S3 step, use
The sequencing of Sanger method.
9. the method for HLA-B High Resolution Gene Typing according to claim 7 or 8, which is characterized in that adopt in the S3 step
Nucleotide sequence with the sequencing primer of Sanger method includes following sequence:
(c1) sequence is as shown in SEQ ID NO.5-SEQ ID NO.10.
10. the method for HLA-B High Resolution Gene Typing according to claim 9, which is characterized in that in the S3 step,
Amplification reaction system is sequenced in Sanger method, is to be calculated as with 10 μ L:
Sequencing primer, 5 μM, 0.75 μ l；
Sequencing template DNA, 2.0 μ l；
BigDye Terminator v3.1 Ready Reaction Mix (ABI), 0.25 μ l
Sequencing buffer, 3.0 μ l；
Add ddH2O to 10 μ l.
11. the method for HLA-B High Resolution Gene Typing according to claim 7 or 8, which is characterized in that in the S3 step,
It is as follows that amplification program is sequenced in Sanger method:
96 DEG C of initial denaturation 1min；96 DEG C of holdings 10Sec, 60 DEG C of holding 2min react 40 circulations；10 DEG C of preservations.
12. a kind of kit for HLA-B High Resolution Gene Typing, which is characterized in that including of any of claims 1 or 2
The kit of HLA-B gene magnification is used for described in primer sets and/or claim 3 or 4 for HLA-B gene magnification.
13. the kit according to claim 12 for HLA-B High Resolution Gene Typing, which is characterized in that including surveying
Sequence amplification reaction system is to be calculated as with 10 μ L:
Sequencing primer, 5 μM, 0.75 μ l；
Sequencing template DNA, 2.0 μ l；
BigDye Terminator v3.1 Ready Reaction Mix (ABI), 0.25 μ l；
Sequencing buffer, 3.0 μ l；
Add ddH2O to 10 μ l；
The sequencing primer is as shown in SEQ ID NO.5-SEQ ID NO.10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810333623.9A CN108531568B (en) | 2018-04-13 | 2018-04-13 | It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810333623.9A CN108531568B (en) | 2018-04-13 | 2018-04-13 | It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108531568A CN108531568A (en) | 2018-09-14 |
| CN108531568B true CN108531568B (en) | 2019-09-13 |
Family
ID=63480026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810333623.9A Active CN108531568B (en) | 2018-04-13 | 2018-04-13 | It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108531568B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101654691A (en) * | 2009-09-23 | 2010-02-24 | 深圳华大基因科技有限公司 | Method for amplifying and typing HLA gene and relevant primer thereof |
| CN101892317A (en) * | 2010-07-29 | 2010-11-24 | 苏州大学 | HLA high-resolution gene sequencing kit |
| CN101962676A (en) * | 2010-08-31 | 2011-02-02 | 深圳市血液中心 | Human leukocyte antigen HLA-A and HLA-B gene full-length sequencing method and HLA gene sequencing and typing method |
| CN105039332A (en) * | 2015-08-19 | 2015-11-11 | 苏静 | Group-specific amplification primer, sequence-based typing method and kit for HLA genes |
-
2018
- 2018-04-13 CN CN201810333623.9A patent/CN108531568B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101654691A (en) * | 2009-09-23 | 2010-02-24 | 深圳华大基因科技有限公司 | Method for amplifying and typing HLA gene and relevant primer thereof |
| CN101892317A (en) * | 2010-07-29 | 2010-11-24 | 苏州大学 | HLA high-resolution gene sequencing kit |
| CN101962676A (en) * | 2010-08-31 | 2011-02-02 | 深圳市血液中心 | Human leukocyte antigen HLA-A and HLA-B gene full-length sequencing method and HLA gene sequencing and typing method |
| CN105039332A (en) * | 2015-08-19 | 2015-11-11 | 苏静 | Group-specific amplification primer, sequence-based typing method and kit for HLA genes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108531568A (en) | 2018-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988353B (en) | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit | |
| CN111996265B (en) | SNP molecular marker influencing wool fiber diameter of fine wool sheep and application thereof | |
| JP2013529472A (en) | Application of PCR sequencing method based on DNA molecular tag technology and DNA incomplete fragmentation technology in HLA genotyping | |
| CN101928776A (en) | PCR-SBT method for HLA genotyping and reagent thereof | |
| CN112048562B (en) | SNP molecular marker influencing diameter of alpine merino sheep wool fiber and application thereof | |
| CN113913530B (en) | Molecular marker related to sheep body height and application thereof | |
| CN104894230B (en) | The group-specific primers PCR-SBT methods and reagent of a kind of HLA-DQB1 Genotypings | |
| CN109234442A (en) | One kind SNP marker relevant to the more lamb characters of sheep and its detection kit and application | |
| CN114959064A (en) | Molecular detection method for identifying recessive white feather genotype of chicken by anticoagulation whole blood method and application thereof | |
| CN108913787A (en) | SNP marker relevant to the more lambs of sheep and its application | |
| CN108588205B (en) | It is a kind of for the primer sets of HLA-DRB1 High Resolution Gene Typing, kit and method | |
| CN107937487B (en) | It is a kind of for the amplification of HLA-A gene PCR, the method for Genotyping, primer sets and kit | |
| CN113604580A (en) | Primer and kit for identifying rose hip genotype by whole blood method and application | |
| CN108165635B (en) | KIAA1462 gene promoter region variant site and application thereof in improving egg laying performance of geese | |
| CN108531568B (en) | It is a kind of for HLA-B gene magnification, the primer sets of Genotyping, kit and method | |
| TW201300528A (en) | Method for hla-dqb1 genotyping and related primers thereof | |
| CN108441547A (en) | A kind of HLA gene magnifications, the primer sets of Genotyping, kit and method | |
| CN105861675A (en) | Primers, probe, kit and method for microchimerism detection and individual recognition | |
| CN112609006B (en) | Human leukocyte antigen one-step sequencing and typing method and application thereof | |
| CN106755422B (en) | Detection method of MEG3 gene SNP related to cattle growth traits and application thereof | |
| CN114854737A (en) | Class I HLA gene amplification primer, kit and typing method based on third-generation sequencing platform | |
| CN107586856A (en) | A kind of Rhinopithecus roxellana Paternity and individual identification method based on SNP site | |
| CN112852949A (en) | Molecular marker of Kazakh EH, primer pair and application thereof | |
| CN109355366A (en) | HLA-B high-resolution gene sequencing kit | |
| CN109852682A (en) | HLA-A allele sequencing and typing kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |