CN101928770B - Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene - Google Patents
Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene Download PDFInfo
- Publication number
- CN101928770B CN101928770B CN2009101904784A CN200910190478A CN101928770B CN 101928770 B CN101928770 B CN 101928770B CN 2009101904784 A CN2009101904784 A CN 2009101904784A CN 200910190478 A CN200910190478 A CN 200910190478A CN 101928770 B CN101928770 B CN 101928770B
- Authority
- CN
- China
- Prior art keywords
- exon
- primer
- sequence
- hla
- sequencing
- 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.)
- Expired - Fee Related
Links
Images
Abstract
The invention provides a sequencing-based typing method of human leucocyte antigen (HLA)-Cw genes, comprising the following steps: a. carrying out PCR amplification on a typing target region by two pairs of PCR amplification primers, wherein the first pair of amplification primers contains a sequence from a first exon to a fourth exon, and the second pair of amplification primers contains a sequence from a fifth exon to an eighth exon; and b. carrying out sequencing reaction on the obtained amplified product by twelve forward sequencing primers and reverse sequencing primer, wherein the first exon, the second exon, the third exon, the fourth exon and the fifth exon of the HLA-Cw genes are subject to forward and reverse sequencing respectively, the sixth exon is subject to forward sequencing and the seventh exon is subject to reverse sequencing. The invention is established based on full-length sequence of the HLA-Cw genes and single nucleotide polymorphisms (SNPs) data, solving the problems of missed detection of an allele Cw*0706 occurred in population detection via an AlleleSEQR HLA-Cw plus kit and ambiguous results occurred in sequencing-based typing. The method is applicable to high-resolution horizontal gene typing of the HLA-Cw genes in Chinese population, and is beneficial to various application and basic researches of the HLA-Cw genes.
Description
Technical field
Invention relates to biomedicine, particularly relates to the method for the human leucocyte antigen HLA-Cw gene sequencing somatotype of a kind of clinical transplantation tissue matching.
Background technology
The albumen of human leucocyte antigen HLA-Cw genes encoding is classical H LA I quasi-molecule; Not only submission endogenous antigen peptide is given CD8+ T cell recognition; The immunne response of mediation killer T cell, and as killer cell immunoglobulin-like receptor (Killer Immunoglobulin-like Receptor, part KIR); Regulate the killing ability of nk cell (Natural Killer cells), in transplantation immunity, tumour immunity and anti-infectious immunity, play a significant role.The HLA-Cw molecule by a α polypeptied chain (45 kDa) with polymorphum and one by the heterodimer that is positioned at No. 15 genome codings, does not have 2 microglobulins (12 kDa) of polymorphum to form through non covalent bond.
Along with biological function, HLA-Cw gene and the disease association of HLA-Cw molecule and HLA-Cw gene with transplant researchs such as dependency carry out and deep; People are broken traditional wrong understanding of HLA-Cw gene gradually, and HLA-Cw molecule extremely investigator is paid close attention to and paid attention to.
The gene of coding HLA-Cw molecule α chain contains 8 exons, 7 introns altogether.8 exons different peptide domain of encoding respectively, each exon and intron sequences are different in size, and be as shown in table 1:
Table 1Each exon of HLA-Cw gene and intron length
| Exon (Exon) or intron (Intron) | Length (bp) |
| Exon 1 | 73 |
| |
130 |
| Exon 2 | 270 |
| |
251 |
| Exon 3 | 276 |
| |
587 |
| Exon 4 | 276 |
| |
124 |
| Exon 5 | 120 |
| Intron 5 | 441 |
| Exon 6-7-8 | 86 |
| Intron 6 | 107 |
| |
164 |
Like table 1, the signal peptide of a weak point of the 1st exons coding (sequence length 73 bp) instructs the α chain to insert endoplasmic reticulum, and is being excised behind the protein translation, before being expressed on the cytolemma.2nd, 3,4 exons difference coding for alpha
1, α
2, α
3Structural domain, is striden the initial portion of film district and cytoplasmic domain afterbody, the endochylema tailer of the 6th, 7,8 exons coding bands alkalescence anchor residues at the 5th exons coding connection peptides.Peptide-binding groove (peptide-binding cleft) is arranged at top, antigen peptide land, by the α of α chain
1, α
2Two structural domains constitute, and two ends are closed, generally combine the peptide section of 8-11 amino-acid residues, and this groove is the antigen presentation position.Because the important antigen peptide submission structural domain of the 2nd, the 3rd exons coding function; Therefore; Sequencing and typing (the Sequence-based typing of HLA-Cw gene in the past; SBT) method mainly is that the 2nd, 3 exons to the height polymorphum carry out, but the current detection that has generally also increased by the 4th exon polymorphum is promptly carried out sequencing and typing to HLA-Cw gene the 2nd, 3,4 exons, to reduce the equivocal result who occurs in the HLA-Cw gene sequencing somatotype.
With polymerase chain reaction (PCR
)Be HLA sequencing and typing (PCR-SBT) method on basis, be described as the gold standard of international HLA gene type.This method is at first carried out the i.e. amplifying target genes fragment at first of pcr amplification to the exon region that needs somatotype, and the pcr amplification product to purifying carries out sequencing reaction, electrophoresis detection and sequential analysis then.The HLA-Cw gene sequencing somatotype reagent that present domestic each HLA somatotype and clinical tissue are joined in the type work generally depends on import.But the sequencing and typing kit of different manufacturers in the world, the segmental molecular size of amplification HLA-Cw gene purpose is different with strategy.Because commercial kit is comparatively expensive, has limited the large-scale application of HLA-Cw gene sequencing somatotype in population genetics and marrow storehouse.And there are three problems in existing commercial kit:
At first, there is the allelic omission situation of HLA-Cw.Like widely used AlleleSEQR HLA-C plus sequencing and typing kit; Its PCR product has two bands; One band extends to the 3rd intron from the 1st exon, and another band is from the 3rd intron to the 7 introns, but concrete two pairs of PCR primer sequences and position thereof are still unexposed.The applicant had once detected the sample of 5 examples through AlleleSEQR HLA-C plus test kit detected result " unusually " in 620 parts of Han nationality's healthy random individualities; Through molecular cloning and monoploid order-checking; And the PCR primer of design voluntarily checks order again; Confirmed that this 5 routine sample all exists AlleleSEQR HLA-C sequencing and typing kit be used to the to increase PCR primer and the Cw*0706 allelotrope sequence of the 1st exon to the 3 introns not match; Cause in the Chinese population omission of Cw*0706 allelotrope and lose (seeing Chinese Journal of Medical Genetics, 2009 the 5th phases).
Secondly, a large amount of new point mutation come to light to be distributed on the 1st, 5,6,7 exons beyond the 2nd, 3,4 exons and to obtain allelotrope and name.By in July, 2009, (Cw*01,02,03,04,05,06,07,08,12,14,15,16,17,18) have 463 kinds of allelotrope in all 14 subsystems of HLA-Cw.During HLA-Cw gene sequencing somatotype, consistent because of the 2nd, 3,4 exon sequences, will cause occurring in the sequencing and typing equivocal result.In the case; Can be according to different allelotrope combinations; Adopt group-specific primers that the 2nd, 3,4 exon surveyed areas are checked order again, or increase the polymorphum detection of other exon (like exons 1,5,6,7), to solve ambiguous result.But sequencing primer mixed solution of HLA-Cw gene extron 1,5,6,7 (Reaction Mix) and group-specific primers etc. all need be purchased, and are included in the conventional sequencing and typing kit of the commercialization of non-HLA-Cw gene, thereby increased experimental cost.
Once more, although the allelotrope number in HLA-Cw site increases news speed, the HLA-Cw allelotrope number with full length sequence is little very little, particularly lacks allelic full length sequence of Chinese population HLA-Cw and SNPs data.
By the end of in April, 2009; The HLA-Cw allelotrope of announcing in the IMGT/HLA DB (http://www.ebi.ac.uk/imgt/hla/) has 440; Only 49 kinds in the HLA-Cw allelotrope that full length sequence is wherein arranged; Most allelotrope lacks SNPs data and the information that is positioned at other exon and non-coding region, has directly influenced the result of high resolution somatotype.For this reason; The applicant has set up HLA-Cw allelotrope full length sequence molecular cloning and sequence measurement (Chinese Journal of Medical Genetics .2009; 26:258-262); Measure and obtained 35 kinds of common HLA-Cw allelotrope full length sequences in the Chinese population, all submit international GenBank DB and HLA factor NK of The World Health Organization (WHO) to, all obtained international endorsement (seeing table 2); Thereby, important evidence is provided for design and develop the HLA high resolution parting kit that is fit to Chinese colony based on the SNPs information in the Chinese population HLA-Cw allelotrope full length sequence.
The 35 kinds of HLA-Cw allelotrope in table 2 Chinese population and the numbering of full length sequence thereof
Summary of the invention
The present invention is intended to SNP (SNPs) data based on Chinese population HLA-Cw gene genetics basis and full length sequence; Create a kind of method that is suitable for the human leucocyte antigen HLA-Cw gene sequencing somatotype of Chinese population; Chinese population is detected the Cw*0706 allelotrope omission phenomenon that occurs to solve widely used AlleleSEQR HLA-C plus commercial kit; And the ambiguous ramification problem that occurs in the solution sequencing and typing, can be used for the application and the fundamental researchs such as clinical transplantation tissue matching, population genetics, anthropology and theory of evolution of HLA-Cw gene.
For realizing above-mentioned purpose, the present invention provides a kind of method of human leucocyte antigen HLA-Cw gene sequencing somatotype, and this method comprises:
A, at first pcr amplification is carried out in somatotype purpose zone by two pairs of pcr amplification primers, wherein, the sequence of one couple of PCR primer amplification the 1st exon to the 4 exons, the sequence of second pair of PCR primer amplification the 5th exon to the 8 exon;
B, amplified production is carried out sequencing reaction through 12 forwards and reverse sequencing primer; Wherein, The 1st, 2,3,4,5 exons to the HLA-Cw gene carry out forward and reverse two-way order-checking respectively, and the 6th exon is carried out forward order-checking and the 7th exon carries out backward sequencing.
Among the step a; The pcr amplification primer of the 1st to the 4th exon comprises upstream primer C-SBT-F1 and downstream primer C-SBT-R1; Wherein, The sequence of upstream primer C-SBT-F1 is: 5'-CCA ATC AGC GTC TCC GCA GTC-3', and the sequence of downstream primer C-SBT-R1 is: 5'-ACC CCY CAT YCC CCT CCT TAC-3'; The pcr amplification primer of the 5th to the 8th exon comprises upstream primer C-SBT-F2 and downstream primer C-SBT-R2; Wherein, The sequence of upstream primer C-SBT-F2 is: 5'-TTM TCA GRG AAA GCA GAA GTC-3', the sequence of downstream primer C-SBT-R2 is: 5'-AAT CCT GCA TCT CAG TCC CAC-3'.
The upstream primer C-SBT-F1 of the pcr amplification primer of the 1st to the 4th exon is positioned at 5 '-promoter region of HLA-Cw gene; Downstream primer C-SBT-R1 is positioned at the 4th intron zone of HLA-Cw gene; Amplification region has contained the 2nd, 3,4 exons; Also comprised part 5 '-promoter region, the 1st exon, the 1st intron, the 2nd intron, the 3rd intron and part the 4th intron sequences, and can increase simultaneously the multiple allelotrope of SNP is arranged at PBR.
The upstream primer C-SBT-F2 of the pcr amplification primer of the 5th to the 8th exon is positioned at the 4th intron zone of HLA-Cw gene; Downstream primer C-SBT-R2 is positioned at 3 '-non-translational region of HLA-Cw gene; The 5th, 6,7,8 exons are contained in the pcr amplification zone, and comprise the sequence in part the 4th intron, the 5th intron, the 6th intron, the 7th intron and part 3 ' UTR zone.
Among the step b, said sequencing primer comprises:
The first exon forward sequencing primer C1F, its sequence is: 5 '-GTTCTRAAGTCCCCAGTC-3 ', the first exon reverse sequencing primer C1R, its sequence is: 5 '-GAAATACYTCATGGAGTG-3 ';
The second exon forward sequencing primer C2F, its sequence is: 5'-GGGTCTCAGCCMCTCCTC-3', the second exon reverse sequencing primer C2R, its sequence is: 5'-GCC GTC CGT GGG GGA TG-3';
The 3rd exon forward sequencing primer C3F, its sequence is: 5'-GCCCCAGTCRCCTTTAC-3', the 3rd exon reverse sequencing primer C3R, its sequence is: 5'-TTCCTCCCCTCCTCGTG-3';
The 4th exon forward sequencing primer C4F, its sequence is: 5'-TTCTCAGGATGGTCACATG-3', the 4th exon reverse sequencing primer C4R, its sequence is: 5'-CCYCATYCCCCTCCTTAC-3';
The 5th exon forward sequencing primer C5F, its sequence is: GTCAGGGCTGAGGCTTG, the 5th exon reverse sequencing primer C5R, its sequence is: GATGGTGCTTCCAGTAAC;
The 6th exon forward sequencing primer C6F, its sequence is: TCCAAGACTAGGAGGTTC, the 7th exon reverse sequencing primer C7R, its sequence is: CAGGTCAGTCATGGTAAG.
The amplified fragments length of the pcr amplification primer of the 1st to the 4th exon is 1960 bp.
The pcr amplification primer expanding fragment length of the 5th to the 8th exon is 1050 bp.
The pcr amplification reaction system of the 1st to the 4th exon consists of:
10×Pfu?Buffer 2.5 L
dNTP（25?mM） 2.0 L
Each 1 L of PCR primer (10 μ M)
Genomic dna 100 ng
Pfu enzyme (5 U/ L) 0.5 L
Glycerine (50%) 1.0 L
Add ddH
2O to 25 L;
The pcr amplification reaction system of the 5th to the 8th exon consists of:
2×GC?Buffer 12.5 L
dNTP（25?mM） 1.0 L
Each 1 L of PCR primer (10 μ M)
Genomic dna 100 ng
LA Taq enzyme (5 U/ L) 0.5 L
MgCl
2?(25mM) 1.5 L
Add ddH
2O to 25 L
The pcr amplification of two pairs of PCR primers carries out through the PCR appearance, and its loop parameter is:
95℃ 3?min
95℃ 30?Sec
62℃ 30?Sec
72 ℃ of 2 min, 30 circulations
72℃ 15?min
4℃ Infinite
The condition of sequencing reaction is:
Sequencing primer (1.0 μ M) 3.2 L
Sequencing template DNA 2.0 L
ABI?PRISM?BigDye?Terminator?1.1 1.0 l
Add ddH
2O to 10 L
The present invention is based on the human leucocyte antigen HLA-Cw full length gene sequence of Chinese population; Created a kind of method of the HLA-Cw of being used for gene sequencing somatotype; Its contribution is; It efficiently solves widely used AlleleSEQR HLA-C plus commercial kit Chinese population is detected the Cw*0706 allelotrope omission phenomenon that occurs, and solves the ambiguous ramification problem that occurs in the sequencing and typing.The present invention is through four locus specificity pcr amplification primers and 12 sequencing primers, and exploration optimum annealing temperature and adjusting Mg
2+Condition such as ion and primer concentration can be carried out PCR specific amplification and sequencing and typing to the HLA-Cw gene.No background signal and Za Feng in the sequence that is obtained are easy to identification and interpretation as a result.Because during design of primers; Taken into full account the HLA-Cw allelotrope SNP (SNPs) of PBR, thereby can solve HLA-Cw allelotrope (like the Cw*0706) omission that occurs when the import reagent box detects the Chinese population dna sample effectively and lose phenomenon.This method also can be carried out the 1st, 5,6,7 exon polymorphums and detect except can being carried out the conventional sequencing and typing of HLA-Cw gene the 2nd, 3,4 exons, reduces ambiguous result; This method is suitable for the high-resolution level gene type of Chinese population HLA-Cw gene, and the application and the basic research works such as clinical transplantation tissue matching, population genetics, anthropology and theory of evolution of HLA-Cw gene.
Description of drawings
Fig. 1 is that 5 examples of embodiment 1 use the import reagent box to detect the 1st to the 4 exon amplification reaction effect figure that doubts to Cw*0706 omission sample.
Fig. 2 is the routine sample of embodiment 1 detects institute's calling sequence and result respectively with an Atria AlleleSEQR HLA-C plus test kit and detection method of the present invention contrast synoptic diagram; Wherein, Fig. 2 A is a relatively synoptic diagram of the 2nd exon sequencing result; Fig. 2 B is a relatively synoptic diagram of the 3rd exon sequencing result, and Fig. 2 C is a relatively synoptic diagram of the 4th exon sequencing result.
Fig. 3 is twice expanding effect figure of sample among the embodiment 2, and wherein, Fig. 3 A is the 1st to the 4th exon amplification design sketch, and Fig. 3 B is the 5th to the 8th exon amplification design sketch.
Fig. 4 is the HLA-Cw gene the 1st (A) of marrow aspiration donor sample, 2 (B), 3 (C), 4 (D), 5 (E), the forward and reverse primer sequencing result of 6 and 7 (F) exon synoptic diagram.
Fig. 5 is that all sequences imports in Assign 3.5 SBT (Conexio Genomics, the Western Australia) software and can clear interpretation go out person under inspection's genotype synoptic diagram.
Embodiment
The following example is to further explanation of the present invention and explanation, and the present invention is not constituted any limitation.
The molecular genetics that the present invention is based on Chinese population HLA-Cw gene is basic, and the method that provides a kind of HLA-Cw high resolution somatotype to detect is through sequencing primer, exploration optimum annealing temperature and the adjusting Mg of design HLA-Cw gene PCR primer and each exon
2+Condition such as ion and primer concentration can be carried out the high-resolution level sequencing and typing to the HLA-Cw gene.
Totally 8 pairs 16 of dna primer sequences of the present invention entrust the precious biotech firm in Dalian synthetic.The Pfu high-fidelity enzyme that the amplification enzyme adopts Stratagene company to produce, the accuracy of assurance sequence.
The method that present embodiment has provided through human leucocyte antigen HLA-Cw gene sequencing somatotype of the present invention solves the scheme of AlleleSEQR HLA-C plus import reagent box to Cw*0706 allelotrope omission problem.
AlleleSEQR HLA-C plus test kit, its PCR product has two bands, and a band extends to the 3rd intron from the 1st exon, and another band is from the 3rd intron to the 7 introns.The individual sample of 620 routine Han nationality healthy random detects through AlleleSEQR HLA-C plus test kit; We have found that 5 routine samples do not have with it the genotype of coupling fully; All there be not matching of single base with immediate multiple allelotype; And the base heterozygosis occurs at the 4th exon, prompt for heterozygote, but in the 2nd, the 3rd exon region of rich polymorphism no heterozygosis base.
At first; get the upstream primer C-SBT-F1 and the downstream primer C-SBT-R1 of the one couple of PCR primers among the present invention, the 1st to the 4th exon of amplification HLA-Cw gene, carry out pcr amplification to 5 routine exceptional samples; increase and carry out in ABI 9700 type PCR appearance, amplification reaction system consists of:
10×Pfu?Buffer 2.5 L
dNTP（25?mM） 2.0 L
Each 1 L of PCR primer (10 μ M)
Genomic dna 100 ng
Pfu enzyme (5 U/ L) 0.5 L
Glycerine (50%) 1.0 L
Add ddH
2O to 25 L
The amplification cycles parameter is:
95℃ 3?min
95℃ 30?Sec
62℃ 30?Sec
72 ℃ of 2 min, 30 circulations
72℃ 15?min
4℃ Infinite
Get 5 L PCR products, through EB dyeing, electrophoresis in 1% sepharose; Contrast DL2000 dna molecular marker is observed specific PCR product band in gel imaging system.PCR product band is clear, special, sees Fig. 1.
In remaining pcr amplification product, add 4 L ExoSAP-IT enzymes and handle, to remove unnecessary PCR primer and substrate dNTPs.
Then, get forward, reverse sequencing primer C2F, the C2R of the 2nd among the present invention, 3,4 exons; C3F, C3R; C4F, C4R carry out two-way order-checking, and its sequencing reaction system is formed as follows:
Sequencing primer (1.0 μ M) 3.2 L,
Sequencing template DNA 2.0 L,
ABI?PRISM?BigDye?Terminator?1.1 1.0 l，
Add ddH
2O to 10 L
The sequencing reaction program is:
98℃ 2?min
96℃ 10?Sec
50℃ 5?Sec
60 ℃ of 4 min, 30 circulations
4℃ Infinite
After sequencing reaction is accomplished; Electrophoresis in ABI 3730 sequenators; The result that the result of derived sequence analysis and Atria AlleleSEQR HLA-C plus import reagent box draw compares; Find: this 5 routine sample that Atria AlleleSEQR test kit detects, there is not with it the genotype of coupling fully, the sequence of HLA-Cw gene the 2nd, the 3rd exon all is the base peak that isozygotys.And, then obtained the genotype of coupling fully by after one couple of PCR primer amplification of the present invention and the order-checking, do not detect new allelotrope; And at the 2nd, 3 exons heterozygosis base peak has appearred respectively.Result's contrast shows: these 5 are all carried the allelic sample of Cw*0706, detect through AlleleSEQR HLA-C plus test kit all to have the omission of Cw*0706 allelotrope and lose phenomenon.
As shown in Figure 2, a sample (genotype: Cw*0802,0706) detects through AlleleSEQR HLA-C plus test kit, and the 2nd, 3 exons are the base of isozygotying, and have only the sequence of Cw*0802.And the sequencing result of method of the present invention shows, the heterozygosis base all appears in the 2nd, 3,4 exons of 5 routine samples, has obtained the genotype of coupling (full match) fully, does not find new base point mutation.Result's contrast shows: this 5 routine sample detects through AlleleSEQR HLA-C plus test kit and all has the omission of Cw*0706 allelotrope and lose phenomenon.
Present embodiment provides the instance that 156 routine marrow aspiration donor samples is carried out HLA-Cw high resolution sequencing and typing.
The applicant has selected 156 routine samples from the marrow aspiration donor in Shenzhen, carry out sequencing and typing with PCR primer among the present invention and sequencing primer, verifies implementation result of the present invention.Selected 156 routine samples have comprised all subsystems of present HLA-Cw.With amplification among the present invention and sequencing primer above-mentioned sample is carried out the sequencing and typing experiment, all obtained the HLA-Cw genotype at last.
At first through two pairs of pcr amplification primers to sample is carried out pcr amplification reaction; Wherein, First pair of amplimer comprises from the sequence of the 1st exon to the 4 exons, and second pair of amplimer comprises from the sequence of the 5th exon to the 8 exons, and two amplified reaction results see Fig. 3.
Amplification reaction condition: each sample is provided with two kinds of amplification reaction systems, and wherein the amplification reaction system of one couple of PCR primers is formed as follows:
10×Pfu?Buffer 2.5 L
dNTP（25?mM） 2.0 L
Each 1 L of PCR primer (10 μ M)
Genomic dna 100 ng
Pfu enzyme (5 U/ L) 0.5 L
Glycerine (50%) 1.0 L
Add ddH
2O to 25 L
The amplification reaction system of second pair of PCR primer is formed as follows:
2×GC?Buffer 12.5 L
dNTP（25?mM） 1.0 L
Each 1 L of PCR primer (10 μ M)
Genomic dna 100 ng
LA Taq enzyme (5 U/ L) 0.5 L
MgCl
2?(25mM) 1.5 L
Add ddH
2O to 25 L
After setting the PCR reaction system, in same ABI 9700 type PCR appearance, carry out synchronous amplification, loop parameter is following:
95℃ 3?min
95℃ 30?Sec
62℃ 30?Sec
72 ℃ of 2 min, 30 circulations
72℃ 15?min
4℃ Infinite
After amplified reaction finishes, with 12 sequencing primers, i.e. the forward and reverse sequencing primer C1F of first exon, C1R; The forward and reverse sequencing primer C2F of second exon, C2R; The forward and reverse sequencing primer C3F of the 3rd exon, C3R; The forward and reverse sequencing primer C4F of the 4th exon, C4R; The forward and reverse sequencing primer C5F of the 5th exon, C5R; The 6th exon forward sequencing primer C6F; The 7th exon reverse sequencing primer C7R carries out sequencing reaction to corresponding pcr template, can find out that the base fignal center of HLA-Cw gene the 1st, 2,3,4,5,6,7 exon forwards and/or backward sequencing is higher, no background signal and Za Feng in the sequence.
Sequencing result shows that in 156 increments basis, the result of (full match) is mated in 155 these appearance of increment fully, another increment genotype and Cw*010201 originally, and 010201 is the most approaching, but exist single base not match at the 3rd exon.Further adopt one couple of PCR primers of the present invention to amplification HLA-Cw gene extron 1～4; Pcr amplification product is carried out molecular cloning and monoploid order-checking; Confirm that this sample carries the relevant neomorph of Cw*01; The difference of this neomorph and immediate Cw*010201 is the 3rd exon: genome sequence nt 959 T>and C (codon 171 TAC>CAC) the missense mutation (see figure 4), cause an amino-acid residue to replace (Tyr171His).This sequence has been submitted GenBank to, and (number of registration: GQ373181) with WHO HLA factor NK (HWS10006611), and by definite designation is Cw*0130.
In all sequences importing Assign 3.5 SBT (Conexio Genomics, Western Australia) software, clearly interpretation goes out person under inspection's genotype, and is as shown in Figure 5:
SEQUENCE?LISTING
< 110>Shenzhen Blood Center
< 120>method of human leucocyte antigen HLA-Cw gene sequencing somatotype
<140>?200910190478.4
<141>?2009-09-18
<160>?16
<210>?1
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
The pcr amplification upstream primer C-SBT-F1 of the < 223>the 1st to the 4th exon
<400>?1
ccaatcagcg?tctccgcagt?c?21
<210>?2
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
The pcr amplification downstream primer C-SBT-R1 of the < 223>the 1st to the 4th exon
<400>?2
accccycaty?cccctcctta?c?21
<210>?3
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
The pcr amplification upstream primer C-SBT-F2 of the < 223>the 5th to the 8th exon
<400>?3
ttmtcagrga?aagcagaagt?c?21
<210>?4
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
The pcr amplification downstream primer C-SBT-R2 of the < 223>the 5th to the 8th exon
<400>?4
aatcctgcat?ctcagtccca?c?21
<210>?5
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>first exon forward sequencing primer C1F
<400>?5
gttctraagt?ccccagtc?18
<210>?6
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>first exon reverse sequencing primer C1R
<400>?6
gaaatacytc?atggagtg?18
<210>?7
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>second exon forward sequencing primer C2F
<400>?7
gggtctcagc?cmctcctc?18
<210>?8
<211>?17
<212>?DNA
< 213>artificial sequence
<220>
< 223>second exon reverse sequencing primer C2R
<400>?8
gccgtccgtg?ggggatg?17
<210>?9
<211>?17
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 3rd exon forward sequencing primer C3F
<400>?9
gccccagtcr?cctttac?17
<210>?10
<211>?17
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 3rd exon reverse sequencing primer C3R
<400>?10
ttcctcccct?cctcgtg?17
<210>?11
<211>?19
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 4th exon forward sequencing primer C4F
<400>?11
ttctcaggat?ggtcacatg?19
<210>?12
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 4th exon reverse sequencing primer C4R
<400>?12
ccycatyccc?ctccttac?18
<210>?13
<211>?17
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 5th exon forward sequencing primer C5F
<400>?13
gtcagggctg?aggcttg?17
<210>?14
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 5th exon reverse sequencing primer C5R
<400>?14
gatggtgctt?ccagtaac?18
<210>?15
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 6th exon forward sequencing primer C6F
<400>?15
tccaagacta?ggaggttc?18
<210>?16
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the 7th exon reverse sequencing primer C7R
<400>?16
caggtcagtc?atggtaag?18
Claims (5)
1. the method for a human leucocyte antigen HLA-Cw gene sequencing somatotype is characterized in that this method comprises:
A, at first pcr amplification is carried out in somatotype purpose zone by two pairs of pcr amplification primers; Wherein, The sequence of one couple of PCR primer amplification the 1st exon to the 4 exons; The sequence of second pair of PCR primer amplification the 5th exon to the 8 exon, the pcr amplification primer of said the 1st to the 4th exon comprises upstream primer C-SBT-F1 and downstream primer C-SBT-R1, wherein; The sequence of upstream primer C-SBT-F1 is: 5'-CCA ATC AGC GTC TCC GCA GTC-3', and the sequence of downstream primer C-SBT-R1 is: 5'-ACC CCY CAT YCC CCT CCT TAC-3'; The pcr amplification primer of the 5th to the 8th exon comprises upstream primer C-SBT-F2 and downstream primer C-SBT-R2; Wherein, The sequence of upstream primer C-SBT-F2 is: 5'-TTM TCA GRG AAA GCA GAA GTC-3', and the sequence of downstream primer C-SBT-R2 is: 5'-AAT CCT GCA TCT CAG TCC CAC-3';
B, amplified production is carried out sequencing reaction through 12 forwards and reverse sequencing primer; Wherein, The 1st, 2,3,4,5 exons to the HLA-Cw gene carry out forward and reverse two-way order-checking respectively; The 6th exon is carried out forward order-checking and the 7th exon carries out backward sequencing, and said sequencing primer comprises:
The first exon forward sequencing primer C1F, its sequence is: 5 '-GTTCTRAAGTCCCCAGTC-3 ', the first exon reverse sequencing primer C1R, its sequence is: 5 '-GAAATACYTCATGGAGTG-3 ';
The second exon forward sequencing primer C2F, its sequence is: 5'-GGGTCTCAGCCMCTCCTC-3', the second exon reverse sequencing primer C2R, its sequence is: 5'-GCC GTC CGT GGG GGA TG-3';
The 3rd exon forward sequencing primer C3F, its sequence is: 5'-GCCCCAGTCRCCTTTAC-3', the 3rd exon reverse sequencing primer C3R, its sequence is: 5'-TTCCTCCCCTCCTCGTG-3';
The 4th exon forward sequencing primer C4F, its sequence is: 5'-TTCTCAGGATGGTCACATG-3', the 4th exon reverse sequencing primer C4R, its sequence is: 5'-CCYCATYCCCCTCCTTAC-3';
The 5th exon forward sequencing primer C5F, its sequence is: GTCAGGGCTGAGGCTTG, the 5th exon reverse sequencing primer C5R, its sequence is: GATGGTGCTTCCAGTAAC;
The 6th exon forward sequencing primer C6F, its sequence is: TCCAAGACTAGGAGGTTC, the 7th exon reverse sequencing primer C7R, its sequence is: CAGGTCAGTCATGGTAAG.
2. the method for claim 1; It is characterized in that; The upstream primer C-SBT-F1 of the pcr amplification primer of said the 1st to the 4th exon is positioned at 5 '-promoter region of HLA-Cw gene; Downstream primer C-SBT-R1 is positioned at the 4th intron zone of HLA-Cw gene; Amplification region has contained the 2nd, 3,4 exons, has also comprised part 5 '-promoter region, the 1st exon, the 1st intron, the 2nd intron, the 3rd intron and part the 4th intron sequences, and can increase simultaneously the multiple allelotrope of SNP is arranged at PBR.
3. the method for claim 1; It is characterized in that; The upstream primer C-SBT-F2 of the pcr amplification primer of said the 5th to the 8th exon is positioned at the 4th intron zone of HLA-Cw gene; Downstream primer C-SBT-R2 is positioned at 3 '-non-translational region of HLA-Cw gene, and the 5th, 6,7,8 exons are contained in the pcr amplification zone, and comprises the sequence in part the 4th intron, the 5th intron, the 6th intron, the 7th intron and part 3 ' UTR zone.
4. method as claimed in claim 2 is characterized in that, the amplified fragments length of the pcr amplification primer of said the 1st to the 4th exon is 1960 bp.
5. method as claimed in claim 3 is characterized in that, the pcr amplification primer expanding fragment length of said the 5th to the 8th exon is 1050 bp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101904784A CN101928770B (en) | 2009-09-18 | 2009-09-18 | Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101904784A CN101928770B (en) | 2009-09-18 | 2009-09-18 | Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101928770A CN101928770A (en) | 2010-12-29 |
| CN101928770B true CN101928770B (en) | 2012-02-29 |
Family
ID=43368199
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101904784A Expired - Fee Related CN101928770B (en) | 2009-09-18 | 2009-09-18 | Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101928770B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2014355369B2 (en) * | 2013-11-27 | 2020-08-13 | Genodive Pharma Inc. | Simple method and kit for DNA profiling of HLA genes by high-throughput massively parallel sequencer |
| CN105296618A (en) * | 2015-10-14 | 2016-02-03 | 深圳市血液中心 | Human leucocyte antigen genotyping method and reagent thereof |
| CN108796050A (en) * | 2018-06-08 | 2018-11-13 | 深圳市血液中心 | HLA-C1/C2 gene PCRs-SSP rapid identification methods in liver transfer operation |
| WO2020199127A1 (en) * | 2019-04-02 | 2020-10-08 | 中国热带农业科学院热带生物技术研究所 | Design of sequencing primers and pcr-based method for sequencing whole genome |
| CN112662750B (en) * | 2021-01-29 | 2024-01-09 | 深圳市血液中心(深圳市输血医学研究所) | Related primer, kit and method for determining-26-bit base of HLA-E gene regulatory region of human leukocyte antigen |
| CN112746098A (en) * | 2021-02-22 | 2021-05-04 | 深圳荻硕贝肯精准医学有限公司 | KIR2DL2 genotyping kit and genotyping method |
-
2009
- 2009-09-18 CN CN2009101904784A patent/CN101928770B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| L. Delfino等.HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1–5 by sequence specific primers.《Tissue Anligens》.1998,第52卷(第3期),251-259. * |
| 蓝欲晓等.HLA-Cw基因测序分型的研究.《江西医学检验》.2006,第24卷(第4期),306-308. * |
| 邓志辉等.HLA-Cw基因全长序列分子克隆及测序方法的建立.《中华医学遗传学杂》.2009,第26卷(第3期),258-262. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101928770A (en) | 2010-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Campbell et al. | African genetic diversity: implications for human demographic history, modern human origins, and complex disease mapping | |
| CN101928770B (en) | Sequencing-based typing method of human leucocyte antigen (HLA)-Cw gene | |
| CN109468315B (en) | Rice flooding-resistant gene Sub1 codominant molecular marker and application thereof | |
| EP3789506A1 (en) | Prunus mume pendulous trait snp molecular markers and use thereof | |
| CN104059963B (en) | Detection method of Chinese simmental cattle carcass and meat quality trait genetic markers | |
| CN114686597B (en) | SNP molecular marker for sex identification of silver dragon fish and application thereof | |
| Keating et al. | A note on the evaluation of a beta-casein variant in bovine breeds by allele-specific PCR and relevance to β-casomorphin | |
| Guelsin et al. | Genetic polymorphisms of Rh, Kell, Duffy and Kidd systems in a population from the State of Paraná, southern Brazil | |
| Le et al. | Development of a simultaneous high resolution typing method for three SLA class II genes, SLA-DQA, SLA-DQB1, and SLA-DRB1 and the analysis of SLA class II haplotypes | |
| Farhadian et al. | Polymorphisms in the ovine myostatin gene are associated with birth weight but not with weight gain in Iranian Makoei sheep | |
| Svensson et al. | Coat colour and sex identification in horses from Iron Age Sweden | |
| Wu et al. | Restriction fragment length polymorphism in the exon 2 of the BoLA-DRB3 gene in Chinese Holstein of the south China | |
| Rafia et al. | Sequence variations of mitochondrial DNA displacement-loop in Iranian indigenous sheep breeds | |
| WO1998003680A1 (en) | Methods for judging the possibility of the onset of bovine leukemia and the resistance thereto | |
| Li et al. | Allelic variations in exon 2 of caprine MHC class II DRB3 gene in Chinese indigenous goats | |
| Friend et al. | Examination of the role of nitric oxide synthase and renal kallikrein as candidate genes for essential hypertension | |
| Seixas et al. | Sequence diversity at the proximal 14q32. 1 SERPIN subcluster: evidence for natural selection favoring the pseudogenization of SERPINA2 | |
| CN106435006B (en) | The relevant SNP marker of the mouth schizothoracin speed of growth and its application together | |
| CN104531876A (en) | Primer and kit for detecting human APOC3 gene mutation | |
| JP5800307B2 (en) | Method and kit for identifying bovine samples | |
| KR100934438B1 (en) | Genetic marker for estimating carcass weight of beef cattle and the estimation method using the marker | |
| CN110564871B (en) | SNP molecular marker related to yak milk freezing point, primer group, kit and application | |
| Gillett et al. | Characterization of class I–and class II–like major histocompatibility complex loci in pedigrees of north Atlantic right whales | |
| Buhler et al. | High levels of molecular polymorphism at the KIR2DL4 locus in French and Congolese populations: impact for anthropology and clinical studies | |
| McClelland et al. | Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120229 Termination date: 20160918 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |