CN109355366A

CN109355366A - HLA-B high-resolution gene sequencing kit

Info

Publication number: CN109355366A
Application number: CN201811597486.6A
Authority: CN
Inventors: 王林林; 张倩; 周云丽; 刘克瑶
Original assignee: Yinfeng Gene Technology Co Ltd; Yinfeng Biological Group Ltd
Current assignee: Yinfeng Gene Technology Co Ltd; Yinfeng Biological Group Ltd
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2019-02-19

Abstract

The invention discloses HLA-B high-resolution gene sequencing kits, including 1 pair of amplimer, as shown in SEQ ID NO.1,2, and for 5 sequencing primers of sequencing, as shown in NO.3~7 SEQ ID.The invention also discloses the detection methods of HLA-B Genotyping: carrying out PCR amplification using amplimer shown in SEQ ID NO.1,2, is then sequenced using sequencing primer shown in NO.3~7 SEQ ID, so that it is determined that the type of HLA gene.2,3,4 exons are sequenced in kit and method of the invention, can also carry out supplement sequencing, genotyping result combined few to 1,5 exons when necessary.Accurate parting can be carried out to the site B overall length, a generation and two generation microarray datasets can be annexed, main agents voluntarily can configure and optimize in HLA gene sequencing kit, and the reagent cost substantially reduced simultaneously, improves the accuracy rate of parting.

Description

HLA-B high-resolution gene sequencing kit

Technical field

The present invention relates to a kind of HLA-B high-resolution gene sequencing kits, belong to technical field of gene detection.

Background technique

Major histocompatibility complex (MHC) is also known as human leukocyte antigen (HLA), is the something lost of most complex human Pass polymorphism system.HLA gene is located on No. 6 the short arm of a chromosome 6p21.31 of people, the HLA-I class molecule and HLA- of coding II class molecule plays a significant role in regulation immune response, decision disease susceptibility.

According to the function of the product of gene loci and they, HLA can be divided into: class Ⅰ antigens: the production in the site HLA-A, B, C Object, class Ⅱ antigens: the product in the site HLA-DR, DQ, DP, three classes antigen: the complement components such as C4A, C4B, C2, Bf composition.It is polymorphic Property is the important feature of HLA, ends in May, 2018, international immunogenetics database (IMGT, http: // Www.ebi.ac.uk/ipd/imgt/hla/ 18181 kinds of HLA allele) are disclosed, wherein I class and class Ⅱ antigens and transplanting It is closely related.

Accurate HLA typing method and typing data have been widely used in finding in organ and hematopoietic stem cell transplantation Donor, HLA and disease associated research, parenthood determination, individual identification and the disease based on peptide that HLA matches The application fields such as the research and development of poison and cancer vaccine.The degree of cooperation of HLA allele and transplantation effect are closely related between donor-recipient, Accurate HLA parting helps to improve the survival rate after transplanting.

The HLA typing method of international standard has PCR-SSP (sequence specific primers polymerase chain reaction), PCR- at present SSO (hybridization of polymerase chain reaction oligonucleotide probe) and PCR-SBT (sequence-based typing method).The principle of SSO method It is to design the special oligonucleotide sequence of HLA gene type as probe, PCR product is marked, with PCR product and probe Hybridized, judges gene type by detecting signal.SSP method needs to design a whole set of gene-specific primer, passes through PCR Technology obtains specific product, determines gene type by electrophoretic analysis.PCR-SBT is that direct Sequencing analysis base is carried out to DNA Because of classifying method, it is the experimental method for the high-resolution level HLA Genotyping that WHO recommends, DNA sequence dna can be directly obtained, identifies All existing polymorphisms, for most direct, most intuitive method.

Sequencing based type (PCR-SBT), is current HLA high-resolution genotyping " goldstandard ", and SBT method is divided into generation survey Sequence and two generations sequencing (Next Generation sequencing, NGS).PCR-SBT has result accurate and the degree of automation The advantages that high, can be carried out recognition sequence and parting, be even more to have unique advantage in terms of finding new allele.A generation is surveyed It has been a very mature method so far that sequence method was born from 1977, and the sequencing of two generations is sequenced relative to a generation carries out HLA points Type has the characteristics that high throughput, easily automates, reduces equivocal result.Routinely sequencing has the HLA-B gene of generation sequencing at present 2,3,4 exon.

Summary of the invention

For the above-mentioned prior art, the present invention provides a kind of HLA-B high-resolution gene sequencing kits, can answer simultaneously With in generation sequencing and two generations sequencing Illumina platform, in control cost, it is all advantageous to improve typing resolution etc..

The present invention is achieved by the following technical solutions:

HLA-B high-resolution gene sequencing kit, (can including 1 pair of amplimer for expanding HLA-B full length gene 1,2,3,4,5,6,7 exons of HLA-B are expanded, include full exon, amplification length 2.8K), and for HLA-B base to be sequenced Because of 5 sequencing primers of 2,3,4 exons, wherein the nucleotide sequence of 1 pair of amplimer is as follows:

AmpB-F:5'-TGTCGGGACCTTCTTCA-3'；As shown in SEQ ID NO.1；

AmpB-R:5'-AYAGACTCAGCACAGCGAAC-3'；(annexing base Y-TC) is as shown in SEQ ID NO.2；

The nucleotide sequence of 5 sequencing primers is as follows:

SeqB2F:5'-GAGCGCACCGCTGGCG-3'；As shown in SEQ ID NO.3；

SeqB2R:5'-TGGGGTGTCGTGACCTG-3'；As shown in SEQ ID NO.4；

SeqB3F:5'-GGGGGCAGGGACACAC-3'；As shown in SEQ ID NO.5；

SeqB3R:5'-TTTCGTCCTCTTCTCGTTG-3'；As shown in SEQ ID NO.6；

SeqB4F:5'-CTCACGCTCTCACATGG-3'；As shown in SEQ ID NO.7.

Wherein, be sequenced 2 exons primer be seqB2F, seqB2R, sequencing 3 exons sequencing primer be seqB3F, SeqB3R, the sequencing primer of 4 exons of sequencing are seqB4F.

Further, the HLA-B high-resolution gene sequencing kit is made of amplification part and sequencing part, amplification portion Dividing includes following components: PCR buffer (contains Mg²⁺), dNTP Mix, Taq enzyme and amplimer ampB-F and ampB-R；Taq enzyme Select TAKARA LA Taq；Sequencing part includes following components: BigDye v3.1, BigDye v3.1Buffer, sequencing primer SeqB2F, seqB2R, seqB3F, seqB3R, seqB4F.

The detection method of HLA-B Genotyping, comprising the following steps:

(1) sample gene to be tested group DNA is extracted according to routine techniques；

(2) above-mentioned amplimer (ampB-F and ampB-R) is utilized, PCR amplification is carried out to sample to be tested genomic DNA, is obtained To the amplified production containing HLA-B full length gene；

(3) to amplified production electrophoresis: 250V, 10min, and purified using exonuclease and shrimp alkaline phosphotase；

(4) PCR product after purification, using above-mentioned sequencing primer (seqB2F, seqB2R, seqB3F, seqB3R, SeqB4F 2,3,4 exons) are sequenced respectively, obtain sequencing product；

(5) product denaturation, purifying, the sequencing of upper machine will be sequenced, obtain sequencing result；

(6) the exon standard sequence of sequencing result and the HLA gene in database is compared, determines HLA gene Type.

Further, in the step (2) PCR amplification reaction condition are as follows: 96 DEG C, 2min；98 DEG C, 10s → 65 DEG C, 50s → 72 DEG C, 3min (30 circulations)；72 DEG C, 5min；4 DEG C of holdings.

Further, the reaction condition being sequenced in the step (4) are as follows: 96 DEG C, 10s；96 DEG C, 10s → 50 DEG C, 10s → 60 DEG C, 2min (25 circulations)；12 DEG C of holdings.

Basic principle of the invention is: the polymorphism of HLA is complicated, about 5212, HLA-B allele, therefore, to have The PCR primer that can expand the site HLA-B overall length is designed in the conservative region of limit, and for the redesign sequencing of 2,3,4 exons Primer.The design of primers template of HLA-B is B*07:02:01:01 genotype, and allelic sequences are referring to Europe IMGT/HLA Professional website data: http://www.ebi.ac.uk/imgt/hla/index.html.

The technology of the present invention advantage: 2,3,4 exons are sequenced in the present invention, can also carry out when necessary to 1,5 exons Supplement sequencing, genotyping result combined few.Amplification system of the present invention carries out stability verifying in two generation microarray datasets simultaneously, can be right The site B overall length carries out accurate parting, since the present invention can annex a generation and two generation microarray datasets, in HLA gene sequencing kit Main agents voluntarily can configure and optimize, and the reagent cost substantially reduced simultaneously, improves the accuracy rate of parting.

The various terms and phrase that the present invention uses are with well known to a person skilled in the art general senses.

Detailed description of the invention

Fig. 1: amplified sample electrophoretogram.

Peak figure is sequenced in the site Fig. 2: B.

Specific embodiment

Below with reference to embodiment, the present invention is further illustrated.However, the scope of the present invention is not limited to following realities Apply example.One of skill in the art, can be to the present invention it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Carry out various change and modification.

Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..

The design and screening of 1 primer of embodiment

8 pairs of amplimers (as shown in table 1) are devised using Primer Premier5,21 sequencing primers are (such as 2 institute of table Show).

Using the 8 pairs of amplimers designed, to known type, very this is expanded, (concrete operations of amplification refer to electrophoresis Concrete operations in following application examples), and electrophoresis result is observed and recorded, the results are shown in Table 1.As shown in Table 1, first pair, Second pair of amplimer, electrophoretic effects are good (electrophoretic band is single, no disperse), other 6 pairs of amplimers it is ineffective, because This, selects these two pair amplimer to carry out next step experiment.

After obtaining amplified production using above-mentioned two pairs of amplimers, using the sequencing primer in table 2, to 2,3,4 exons Both-end sequencing (concrete operations of survey are with reference to the concrete operations in following application examples) are carried out respectively, obtain sequencing peak figure, observation is simultaneously The superiority and inferiority of record sequencing peak figure, the results are shown in Table 2.As shown in Table 2, in 2 exon forward direction sequencing primers, the 4th article of primer The effect of (shown in SEQ ID NO.3) is best, other 3 primers it is ineffective, therefore, select this primer as the present invention HLA-DQB1 genotyping kit sequencing primer.Similarly, other several sequencing primers are screened, it is final to determine, SEQ ID NO.1, amplimer shown in 2, sequencing primer shown in SEQ ID NO.3,4,5,6,7 is as HLA- of the invention The sequencing primer of B high-resolution gene sequencing kit.

1 amplified production of table

2 sequencing primer of table

Application example carries out the hrr gene parting in the site HLA-B to human blood sample

Specific step is as follows:

(1) it extracts genomic DNA: being stripped according to Tiangeng company genome DNA extraction kit operational manual.

(2) PCR amplification: genomic DNA is expanded using amplimer.Use the site HLA-B amplimer (SEQ ID No.1, SEQ ID No.2) amplification of HLA-B parting is carried out, amplification program and amplification reaction system be as follows:

Pcr amplification reaction condition are as follows: 96 DEG C, 2min；98 DEG C, 10s → 65 DEG C, 50s → 72 DEG C, 3min (30 circulations)； 72 DEG C, 5min；4 DEG C of holdings.

PCR reaction system is 20 μ l, and composition is as shown in table 3.

Table 3

10×LA Buffer(Mg²⁺)	2μl
		dNTP(2.5mM)	1.6μl
F(10pM)	0.5μl
		R(10pM)	0.5μl
ddH2O	13.2μl
		LATaq	0.2μl
DNA(30ng/μl)	2μl

Wherein, 10 × LA Buffer (Mg²⁺), dNTP (2.5mM), LATaq archaeal dna polymerase be purchased from Bao Sheng biotech firm, Primer is purchased from Invitrogen.The electrophoretogram of amplification gained PCR product is as shown in Figure 1, the results showed that obtains target gene fragment.

(3) amplified production is purified using exonuclease and shrimp alkaline phosphotase.

(4) sequencing amplification is carried out using sequencing primer: using sequencing primer (SEQ ID No.3~SEQ ID No.7) point Other that the 2nd of HLA-B amplified production, 3,4 exons are sequenced, sequencing procedure and sequencing reaction system are as follows:

Sequencing reaction condition are as follows: 96 DEG C, 10s；96 DEG C, 10s → 50 DEG C, 10s → 60 DEG C, 2min (25 circulations)；12℃ It keeps.

Sequencing reaction system is 10 μ l, and composition is as shown in table 4.

Table 4

BigDye v3.1	0.25μl
		BigDye v3.1Buffer	1.9μl
Primer (10pM)	1μl
		ddH2O	5.85μl
Amplification purification product	1μl

Wherein, Bigdye v3.1 is purchased from ABI company.

Conventionally sequencing product is purified.

(5) it carries out exon sequencing: machine on sequencing product after purification being sequenced using 3730 sequenator of ABI company, survey Sequence result is as shown in Fig. 2, sequencing peak figure can recognize phenomenon invariably, and peak figure wave mode is uniform, no miscellaneous peak, and heterozygosis peak dispersion rate is 50% Left and right, whole peak figure is clear, leakless type.

Above-described embodiment is provided to those skilled in the art, how to implement and use to be advocated with full disclosure and description Embodiment, rather than for limiting range disclosed herein.Obvious modification will to those skilled in the art Within the scope of the appended claims.

Sequence table

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Yinfeng Bioengineering Group Co., Ltd.

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<141> 2018-12-26

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tggggtgtcg tgacctg 17

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gggggcaggg acacac 16

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Claims

1.HLA-B high-resolution gene sequencing kit, it is characterised in that: including 1 pair of amplification for expanding HLA-B full length gene Primer, and 5 sequencing primers for 2,3,4 exon of HLA-B gene to be sequenced, wherein the nucleotides sequence of 1 pair of amplimer Column are as follows:

AmpB-F:5'-TGTCGGGACCTTCTTCA-3'；As shown in SEQ ID NO.1；

AmpB-R:5'-AYAGACTCAGCACAGCGAAC-3'；As shown in SEQ ID NO.2；

The nucleotide sequence of 5 sequencing primers is as follows:

SeqB2F:5'-GAGCGCACCGCTGGCG-3'；As shown in SEQ ID NO.3；

SeqB2R:5'-TGGGGTGTCGTGACCTG-3'；As shown in SEQ ID NO.4；

SeqB3F:5'-GGGGGCAGGGACACAC-3'；As shown in SEQ ID NO.5；

SeqB3R:5'-TTTCGTCCTCTTCTCGTTG-3'；As shown in SEQ ID NO.6；

SeqB4F:5'-CTCACGCTCTCACATGG-3'；As shown in SEQ ID NO.7.

2. HLA-B high-resolution gene sequencing kit according to claim 1, it is characterised in that: the HLA-B high-resolution Gene sequencing kit is made of amplification part and sequencing part, and amplification part includes following components: PCR buffer (contains Mg² ⁺), dNTP Mix, Taq enzyme and amplimer ampB-F and ampB-R；Taq enzyme selects TAKARA LA Taq；Sequencing part includes Following components: BigDye v3.1, BigDye v3.1Buffer, sequencing primer seqB2F, seqB2R, seqB3F, seqB3R, seqB4F。

3. application of the HLA-B high-resolution gene sequencing kit of any of claims 1 or 2 in detection HLA-B Genotyping.

The detection method of 4.HLA-B Genotyping, it is characterised in that: the following steps are included:

(2) using amplimer shown in SEQ ID NO.1,2, PCR amplification is carried out to sample to be tested genomic DNA, is contained There is the amplified production of HLA-B full length gene；

(4) PCR product after purification, using SEQ ID NO.3~shown in sequencing primer 2,3,4 exons are sequenced respectively, obtain To sequencing product；

(6) the exon standard sequence of sequencing result and the HLA gene in database is compared, determines the type of HLA gene Not.

5. the detection method of HLA-B Genotyping according to claim 4, it is characterised in that: PCR in the step (2) The reaction condition of amplification are as follows: 96 DEG C, 2min；98 DEG C, 10s → 65 DEG C, 50s → 72 DEG C, 3min, 30 circulations；72 DEG C, 5min；4 DEG C keep.

6. the detection method of HLA-B Genotyping according to claim 4, it is characterised in that: sequencing in the step (4) Reaction condition are as follows: 96 DEG C, 10s；96 DEG C, 10s → 50 DEG C, 10s → 60 DEG C, 2min, 25 circulations；12 DEG C of holdings.