CN102321749B - Polymerase chain reaction-sequence based typing (PCR-SBT) method and kit of MHC class I chain-related gene B (MICB) - Google Patents

Abstract

The invention provides a polymerase chain reaction-sequence based typing (PCR-SBT) method and a kit of a MHC class I chain-related gene B (MICB). Through the PCR-SBT method and the kit, MICB locus exons 2-5 can be typed. The PCR-SBT method comprises the following steps of 1, preparing a human genome DNA, 2, carrying amplification of target gene fragments needing to be amplified by PCR amplimers, wherein the target gene fragments comprise MICB exons 2-5 and introns between the MICB exons 2-5, and 3, carrying out amplification of the PCR products obtained by the step 2 with sequencing primers, carrying out sequencing of the amplified PCR products, and comparing the sequencing result and a standard sequence in a data base to confirm a gene typing result. Through the optimal combination of test conditions and the kit with MICB sequencing functions, oligonucleotide sequences of whole MICB locus exons 2-5 and a part of introns can be amplified effectively, and sequencing of the corresponding exons is realized. Therefore, through the PCR-SBT method and the kit, accurate MICB typing is realized.

Description

A kind of PCR-SBT method and test kit of MICB gene type

Technical field

The invention belongs to biology field, relate to amplification and classifying method for human MHC-I class chain related gene B site (MHC class Ichain-related gene B, MICB), and the supporting test kit of described method.

Background technology

MHC-I class chain genes involved (MHC class I chain-related gene, MIC), be positioned at No. 6 karyomit(e) major histocompatibility complexs of people (major histocompatibility complex, MHC) in the gene region, length is 2Mb, and coded protein molecule is the important part that the reactivity acceptor NKG2D of the immunocytes such as NK cell, gamma delta T cells and CD8+T cell identifies.Found at present 7 MIC gene locuss, only MICA, MICB are functional gene, lay respectively at kinetochore 46kb and 140kb position, near HLA-B site three height linkage disequilibrium.The MICB gene has widely polymorphism, has a plurality of genotype.According to the difference of the exon 2 of coding MICB molecule extracellular region～5 gene orders, 31 MICB allelotrope have been found.MICB gene coded protein MICB molecule participates in the process of multi-infection immunity as the NKG2D part, studies show that in a large number the MICB gene pleiomorphism is relevant with the susceptible of various diseases, also is the important factor that causes transplant rejection simultaneously.The somatotype of current MICB gene there is no extensively and popularizes, and without relevant commercially available reagent.

The HLA typing method has been widely used in a plurality of fields on the other hand, joins the aspects such as related, the paternity test of type, HLA and some disease and human genetics such as donor-recipient's histocompatibility before biological function, organ and the bone marrow transplantation of the research of HLA polymorphism, HLA.The HLA typing method is mainly by serological typing technology, cytology typing method, genotyping technique etc.The essence of individual HLA genetic differences is to encode on the gene of its antigen product, so analyze the most accurately method that the genotype of individual HLA is analyzed the HLA type beyond doubt, its accuracy is far above serology and cell blood system type.Reach its maturity take DNA as the HLA genotyping technique on basis at present, and replace gradually serological method and cell blood system type technology.Methods of genotyping has advantages of unique: the blood sample that needs is few, but the sample prolonged preservation, and corresponding somatotype reagent can prepare in a large number, originates unrestricted.Particularly importantly gene type is accurately reliable, good reproducibility, and its somatotype error rate is far below serological method, and genotyping technique is widely used in real work.

The HLA typing method of mark has PCR-SSP (sequence specific primer-oligomerization polymerase chain reaction), PCR-SSO (polymerase chain reaction oligonucleotide probe hybridization) and PCR-SBT (polymerase chain reaction product sequencing based type) in the world at present.

PCR-SSO (sequence specific oligonucleotide): also claim PCR-ASO (allele specific oligonuceotide), hybridize in order to isotropic substance or nonradioactive labeling's probe and the target fragment product of pcr amplification, judge idiotype according to positive spots.Because MICB allelotrope is very many, just to finish the typing analysis operation also very loaded down with trivial details with regard to needing a lot of probes that each DNA sample is repeatedly hybridized (even tens times), so a kind of reverse hybridization (reverse hybridization) that grows up again on this basis.Various probe is fixed on same the film, again with the PCR Product Labeling, with PCR product (gene DNA to be detected) conversely with probe hybridization.So once hybridization can be finished a plurality of allelic analyses.That this method has is highly sensitive, high specificity, need the advantages such as sample size is few, but the hybridization conditions of different probe must strict unified (such as temperature, ionic concn), error easily occurs; Can not detect new allelotrope, test kit needs constantly upgrading; Also bad to some heterozygote resolving power.A lot of genotype contain identical polymorphism, and only because the difference of arrangement mode, so resolving power is not as good as SSP and SBT.The suitable a large amount of and high purity sample of this method, the hybridization band will be as written source recording prolonged preservation (three kinds of effects relatively).

The PCR-SSP:PCR-SSP method is with designing in advance a whole set of allelotrope group-specific primers (sequence specific primer, SSP), obtain the special amplified production of MICB type by round pcr, can determine the MICB type by electrophoresis direct analysis banding pattern, thereby greatly simplify experimental procedure.Advantage is simple, and resolving power can be from low to high, and cost is low.Shortcoming is to be difficult for automatization; Can not detect new allelotrope, test kit needs constantly upgrading.This method is suitable scattered hangs down sample with purity, will bring up again DNA during repeated experiments and must use ultraviolet gel imaging instrument reservation starting material, increase experimental cost (three kinds of effects relatively).PCR-SSP and PCR-SSO all need a large amount of reagent (primer), and can not identify new gene and pseudogene.Can avoid these problems for target sequence exon and intron sequences Fine design primer.

PCR-SBT: with the gene fragment that pcr amplification will be analyzed, then dna sequence dna is analyzed, can directly be obtained genotype.Resolving power is high, can carry out on a large scale, and tolerance range is high, can directly find new allelotrope.Therefore, using the SBT technology, to carry out the hrr gene somatotype be the gene type gold standard of generally acknowledging in the world at present.There is no on the market at present MICB gene type reagent.

Summary of the invention

The purpose of this invention is to provide a kind of MICB gene type the PCR-SBT method and with the supporting test kit of the method, fill up the blank of MICB site somatotype, further improve resolving power and the accuracy of MICB typing method, reduce simultaneously the cost of experiment.

For this reason, the present invention is by the following technical solutions:

A kind of PCR-SBT method of MICB gene type may further comprise the steps:

(1), extracts testing gene group DNA, the intron between the 2-5 exon of the goal gene fragment MICB that the amplification of use pcr amplification primer will be analyzed reaches;

Described amplimer is to being MICB-F and MICB-R

MICB-F：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-R：5’-GGAGATGGGAAAGCTCCTTT-3’；

Described pcr amplification reaction condition is followed successively by:

1)95℃5min；

2) repeat following 19 circulations:

94 ℃ of 45s → 65 ℃ 45s, every circulation reduces by 0.5 ℃ → 72 ℃ 2min;

3) repeat following 13 circulations:

94℃45s→55℃45s→72℃2min；

4)72℃10min；

5) 4 ℃ of maintenances;

Described PCR reaction system is 20 μ l, and it is composed as follows:

The DNA 10 μ l of concentration 4ng/ μ l

1.49 μ l PCR damping fluid, (PCR damping fluid compound method: 10ml PCR damping fluid contains: the 0.8g tromethane, and the 0.22g sulfate of ammoniac, 0.67ml 1M magnesium chloride is with salt acid for adjusting pH to 8.8; )

1.49μl?5mM?dNTP；

0.5 μ l 10 μ M upstream primer MICB-F;

0.5 μ l 10 μ M downstream primer MICB-R;

0.49 μ l 10% bovine serum albumin;

5.32 μ l 28% sucrose/o-cresolsulfonphthalein dyestuff;

0.12 μ l 5U/ μ l TaqDNA polysaccharase;

Add distilled water and supply volume;

(2), use sequencing primer that step (1) gained PCR product is increased, then each sequence that amplifies is checked order, and the standard sequence in sequencing result and the database is compared, thus definite gene type result;

Described 4 oligonucleotide sequencing primers for sequencing analysis;

MICB-CF1：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-CR2：5’-CCCTGTGCTATGGATGCA-3’

MICB-CF3：5’-CAGGAGTCCACCCTTGACAT-3’

MICB-CR4：5’-GGAGATGGGAAAGCTCCTTT-3’。

Described sequencing reaction system is 10 μ l, and it is composed as follows:

2 μ l PCR products;

The 3 μ l Big Dye damping fluid that checks order;

1 μ l Big Dye checks order terminal;

0.25 the sequencing primer of μ l 10 μ M; (using among MICB-CF1, MICB-CR2, MICB-CF3, the MICB-CR4) at every turn

3.75 μ l distilled water;

Described order-checking amplification reaction condition is followed successively by:

1) repeat following 24 circulations:

96℃10s→50℃5s→60℃4min；

2)4℃5min。

The test kit that above-mentioned method is supporting, the primer of the intron between the 2-5 exon of the goal gene fragment MICB that comprising increases will analyze reaches:

MICB-F：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-R：5’-GGAGATGGGAAAGCTCCTTT-3’；

And 4 primers that are used for sequencing analysis:

MICB-CF1：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-CR2：5’-CCCTGTGCTATGGATGCA-3’

MICB-CF3：5’-CAGGAGTCCACCCTTGACAT-3’

MICB-CR4：5’-GGAGATGGGAAAGCTCCTTT-3’。

In the technique scheme, described primer direction all from 5 ' to 3 '; The MICB design template be all allelotrope sequences of MICB*001 all with reference to European IMGT/HLA professional website database: http://www.ebi.ac.uk/imgt/hla/index.html.

Ultimate principle of the present invention is: the polymorphism of MICB is complicated, 31 in the allelotrope site in MICB site, and constantly there is new allelotrope to be found, therefore will be at the limited conserved regions design PCR primer of 2-5 exon in MICB site that can increase, and design again sequencing primer for different exons respectively.

Because technique scheme is used, the present invention compared with prior art has following advantages:

1. the optimum combination of MICB gene sequencing kit of the present invention and test condition, its experiment condition and experimental system are to grope to obtain by great many of experiments, and experimental result has good stability.Can effectively the increase full length DNA fragment of 2-5 exon and intron thereof in MICB site, and it is checked order, resolving power and accuracy to the MICB gene type improved.

Since in the MICB gene sequencing kit of the present invention main agents configure voluntarily and optimize, agents useful for same is experiment reagent commonly used, simultaneously only with synthetic 4 primers (because MICB-CF1 and MICB-F, MICB-CR4 is identical with the MICB-R sequence can be general), and only carry out 4 sequencing reactions and just can carry out high-resolution phenotypic analysis to sample and (minimumly can only carry out sequencing reaction 2 times to MICB-CR2 and MICB-CF1, it has contained all polymorphic sites substantially), more current existing technology (need synthetic many to primer and carry out repeatedly sequencing reaction) greatly reduces the cost of experiment.

3. there is no at present the test kit that detects for the MICB site both at home and abroad, the present invention has novelty.

4. the object that detects in test process of the present invention is Chinese population, and the result who detects through evidence test kit of the present invention can be applicable to the check of Chinese population accurately and reliably.

Description of drawings

Fig. 1 is pcr amplification primer on the MICB structure iron among the embodiment 1, the sequencing primer position view;

Fig. 2 is gained MICB2-5 exon and intron total length pcr amplification product wherein among the embodiment 1;

Fig. 3 is MICB-CF1 order-checking gained sequencer map (the whole polymorphic sites of 2-3 exon of containing the MICB site) among the embodiment 1;

Fig. 4 is MICB-CR4 order-checking gained sequencer map (the whole polymorphic sites of 4-5 exon of containing the MICB site) among the embodiment 1.

Embodiment

The invention will be further described below in conjunction with embodiment, and can not limit the present invention.

Embodiment 1:

Adopt nucleotides sequence tabulate described pcr amplification primer and sequencing primer (position of described primer is as shown in Figure 1), human blood sample is carried out the hrr gene somatotype in MICB site, concrete steps are as follows:

(1) extracts genomic dna

Carry out extracting according to Promaga company genome DNA extraction test kit process specifications.

(2) pcr amplification

Use MICB site amplimer MICB-F and MICB-R to carry out the amplification of MICB somatotype, above-mentioned amplification procedure and amplification reaction system are as follows:

Described pcr amplification reaction condition is:

1.95℃for?5min

2.94 ℃ for 45s → 65 ℃ for 45s (every circulation reduces by 0.5 ℃) → 72 ℃ of for 2min (repeating 19 circulations)

3.94 ℃ for 45s → 72, ℃ for 45s → 55 ℃ for 2min (repeating 13 circulations)

4.72℃for?10min

5.4 ℃ maintenance

Described PCR reaction system is 20 μ l, its table composed as follows:

The DNA 10 μ l of concentration 4ng/ μ l

1.49 μ l PCR damping fluid, (PCR damping fluid compound method: 10ml PCR damping fluid contains: the 0.8g tromethane, and the 0.22g sulfate of ammoniac, 0.67ml 1M magnesium chloride is with salt acid for adjusting pH to 8.8; )

1.49μl?5mM?dNTP；

0.5 μ l 10 μ M upstream primer MICB-F;

0.5 μ l 10 μ M downstream primer MICB-R;

0.49 μ l 10% bovine serum albumin;

5.32 μ l 28% sucrose/o-cresolsulfonphthalein dyestuff;

0.12 μ l 5U/ μ l TaqDNA polysaccharase;

Add distilled water and supply volume;

Wherein dNTPs, Taq archaeal dna polymerase are all available from Beijing Kang Wei century bio tech ltd, and primer is synthetic by the large Gene science company of Shenzhen China.

The electrophorogram of amplification gained PCR product as shown in Figure 2, the result shows and obtains goal gene fragment (being 2-5 exon and the intron full length DNA fragment thereof in MICB site).

According to routine techniques pcr amplification product is carried out purifying.

(3) use the sequencing primer amplification of checking order.

What sequencing reaction adopted is the asymmetric PCR method, different from the regular-PCR method, and its purpose mainly is to be the preparation ssDNA that checks order, and only needing to add a specific sequencing primer, the other end in the reaction is general BigDye end.Use ABI company 3730 sequenators that the PCR product of purifying in the step (2) is checked order, sequencing result such as Fig. 3 to Fig. 4, at first use sequencing primer MICB-CF1, MICB-CR4 to check order respectively, sequencing result has been contained all polymorphic sites of 2-5 exon in MICB site.In order to guarantee that the result accurately and reliably, adopt MICB-CR2 to survey once in the opposite direction of MICB-CF1, MICB-CF3 surveys once in the opposite direction of MICB-CR4, thereby guarantees that each polymorphic site is determined 2 times, reaches accurately and reliably purpose of each polymorphic site.

Above-mentioned order-checking amplification procedure and order-checking amplification reaction system are as follows:

Described sequencing reaction system is 10 μ l, its table composed as follows:

Reaction system

2 μ l PCR products

3μl?Big?Dye?Buffer(ABI)

1μl?Big?Dye?Terminator(ABI)

0.25 μ l sequencing primer (10 μ M)

3.75 μ l distilled water

Described order-checking amplification reaction condition is:

1.96 ℃ for 5s → 60, ℃ for 10s → 50 ℃ for 4min (repeating 24 circulations)

2.4℃for?5min

(4) carry out sequencing reaction.By Fig. 3 and Fig. 4 are analyzed, comparing the MICB loci gene type that shows this sample with the IMGT/HLA database is heterozygote: MICB*00502-MICB*014, and the heterozygosis site is bimodal nested master drawing shape in sequence chart.

Embodiment 2, the international Quality Control of U.S. UCLA university and the experimental result of Chinese Marrow Donor Program data bank Quality Control

Chinese Marrow Donor Program data bank HLA Quality Control sample is to be selected from the common allelotrope combination of Chinese basically, and does not also find undetected phenomenon with reagent of the present invention, and the result is in full accord with contrast agents.The HLA Quality Control sample of U.S. UCLA university has comprised the HLA allelotrope combination of each ethnic group in the world wide, and its result also is consistent with contrast agents.

Claims (1)

1. the PCR-SBT test kit of a MICB gene type is characterized in that, the primer of the intron between the 2-5 exon of the goal gene fragment MICB that comprising increases will analyze reaches:

MICB-F：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-R：5’-GGAGATGGGAAAGCTCCTTT-3’；

And 4 primers that are used for sequencing analysis:

MICB-CF1：5’-GGACAGCAGACCTGTGTGTTA-3’

MICB-CR2：5’-CCCTGTGCTATGGATGCA-3’

MICB-CF3：5’-CAGGAGTCCACCCTTGACAT-3’

MICB-CR4：5’-GGAGATGGGAAAGCTCCTTT-3’。

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