CN101851673A - Method for detecting tagged single-nucleotide polymorphic loci of six immunity-related genes of human - Google Patents
Method for detecting tagged single-nucleotide polymorphic loci of six immunity-related genes of human Download PDFInfo
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Abstract
The invention provides a method for detecting tagged single-nucleotide polymorphic loci of six immunity-related genes of human, which applies a multi-PCR- fluorescent-labeled single base primer extension-tag microarray hybrid genotyping technology in combination with a genome label TMSNP steam genotyping system to detecting 27 tagged single-nucleotide polymorphic loci of 6 genes; specifically, the method comprises extracting the DNA of a genome, selecting the single-nucleotide polymorphic loci of a candidate gene, designing and synthesizing a single-nucleotide polymorphic locus amplimer and an extension primer, verifying the quality of the single-nucleotide polymorphic locus amplimer by a single PCR, preparing and detecting a single-nucleotide polymorphic locus detection sample and carrying out a data analysis and a method stability evaluation. The invention is simple and economical and has the advantages of high throughput, high accuracy, high generality and automatic detection and can be applied in the research on the genetic character of the immunity-related genes in healthy people or the relation between the immunity-related genes and genetic susceptibility of various diseases.
Description
Technical field
The present invention relates to a kind of tagged single-nucleotide polymorphic loci method that detects six immunity-related genes of human, these genes are people's complement receptor 1 type (CR1), Duffy antigen/Chemokine Receptors (DARC), leukocyte differentiation antigen 59 (CD59), leukocyte differentiation antigen 4 (CD4), leukocyte differentiation antigen 8A (CD8A) and leukocyte differentiation antigen 8B 6 genes such as (CD8B), belong to the Medical Molecular Biology field.
Background technology
Studies show that, red corpuscle has immunity to be sticked, directly kills the antigen foreign matter, regulate complement activity, strengthens panimmunity functions such as NK cell function, binding chemotactic factor, the adjusting of participation B cellular immunization, plays a significant role in the natural immunity and acquired immunity.In erythrocyte surface or endochylema, there is the panimmunity related substances, it is of greatest concern wherein to stick the chemokine receptor function of function and Duffy antigen/Chemokine Receptors with the immunity of complement receptor 1 type, and 59 of leukocyte differentiation antigens have restriction people complement system dissolved cell activity, effect that the mediation red corpuscle-the T cell signal transmits.The T cell then is a main cell of realizing the acquired immunity function, its surperficial leukocyte differentiation antigen 4 and leukocyte differentiation antigen 8 combine with major histocompatibility complex II class and I class antigen respectively, can strengthen T cell and antigen presenting cell or target cell bonded degree, play an important role in the signal transduction of T cell proliferation and differentiation, leukocyte differentiation antigen 4 male T cells and leukocyte differentiation antigen 8 male T cells have immunity auxiliary and immunosuppression and direct killing function respectively.Change has taken place in multiple disease in the level of above-mentioned immune molecule, and is relevant closely with disease generation, development and prognosis, but at present the gene of these molecules and the association study of disease genetic susceptibility do not appeared in the newspapers.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) be meant single nucleotide diversity on the karyomit(e) and the dna sequence polymorphism that causes, it has density height, inheritance stability, is easy to characteristics such as automated analysis, be considered to the third generation genetic marker after restriction fragment length polymorphism, microsatellite polymorphism, can be used for aspects such as the assignment of genes gene mapping, polymorphism analysis, is the common counter of study of disease genetic predisposition, medical diagnosis on disease, drug screening etc.Kind of method for detecting single nucleotide polymorphism surplus having developed 20 at present, as dna sequencing, restriction fragment length polymorphism analysis, allele specific oligonucleotide oligonucleotide hybridization etc., but these methods are not suitable on a large scale, automatization detects; Fa Zhan DNA chip technology had satisfied the needs that high-throughput, automatization detect afterwards, with the DNA chip is detection platform, in conjunction with above-mentioned reaction principle, developed multiple method for detecting single nucleotide polymorphism, but there are shortcomings such as false positive rate height, flux be dumb in general mononucleotide polymorphism chip.(Hirschhorn JN such as Hirschhorn, et al.SBE-TAGS:an array-based method for efficientsingle-nucleotide polymorphism genotyping.Proc Natl Acad Sci U S A, 2000,97 (22): 12164-12169) on the basis of single base extension mononucleotide polymorphism chip, set up the DNA chip technology that combines based on single base extension-label microarray hybridization, dexterously with allele-specific primers sequence and being integrated on the single-basic extension primer with sequence specific bonded sequence label on the chip, the high sensitive of single base extension and the advantages such as multiple reaction of high resolving power and label microarray are merged, overcome the shortcoming of other DNA chip technologies, reached high-throughput, pinpoint accuracy, high universalizable, easy, economic effect.
Single-basic extension method (single base primer extension) claims little sequencing, template mediation dyestuff terminator to mix analysis or single nucleotide polymorphism authenticate technology (single-nucleotide polymorphism-identification technology again, SNP-IT), be the method for detecting single nucleotide polymorphism that development is come out on the basis of dideoxy sequencing method.Its principle is: at first design 1 pair of PCR primer and be used for amplifying the fragment that contains the purpose mononucleotide polymorphism site from genomic dna, design 1 and mononucleotide polymorphism site upstream sequence complementary to be measured extension primer again, 1 base complementrity that 3 of this primer ' last base of end is adjacent with the mononucleotide polymorphism site upstream; To extend primer and mix with the PCR product, the ddNTPs that mixes the different fluorescence dyes of mark carries out extension, because the characteristic of ddNTP, when extending 1 base, promptly when the ddNTP of Jia Ruing and mononucleotide polymorphism site base complementrity, reacts i.e. termination; Judge the base type of mononucleotide polymorphism site by detect extending fluorescence types that base sends.This method is based on the order-checking principle, but owing to only need to survey 1 base, its accuracy has substantially exceeded general sequencing reaction, can be rated as the golden standard that single nucleotide polymorphism detects.
The label microarray is a synthetic oligonucleotide strand (label) in advance at the bottom of the plate of glass-chip plate, and having fluorescently-labeled oligonucleotide chain with these label complementary can combine up by hybridization, then its fluorescent mark of scanning detection.Introduce chip technology and help to realize that high-throughput, automatization detect, and adopt the label microarray to make multiple detection become possibility, can utilize the label that is fixed on the chip easily multiple oligonucleotide chain to be made a distinction exactly.
2005, U.S. Beckman company developed GenomeLab based on this principle
TMSNPstream
The heavy gene type of 12-weight/48-system, the micro-array chip that this system adopts is 384 special orifice plates, it at the bottom of the hole flat glass-chip, from several thousand oligonucleotide chains that are widely used in the DNA chip, chosen tens cross hybridization rates minimum, with database in range gene group homology minimum, and can provide the label of very strong signal, be synthesized in advance on the micro-array chip plate.16 or 52 " point " are arranged at the bottom of each orifice plate of 384 orifice plates, comprise 12 or 48 label and 4 Quality Controls that sequence is different, respectively can be for maximum 12 heavy or 48 heavy extensions.Utilize online design of primers instrument Autoprimer design amplification to contain segmental multiple PCR primer of candidate's mononucleotide polymorphism site purpose and corresponding multiple extension primer, every extend 5 of primer ' end be added with chip on sequence label complementary sequence, be used for combining of extension products and label microarray, the result can be read by the Two Colour Fluorescence on the imager.Its somatotype success ratio reaches 96.5%, and rate of accuracy reached to 99% only needs the DNA of 2ng heavily to reach 48 heavy detections with regard to enough carrying out 12, and flux is flexible, and is easy and simple to handle, the level of automation height.
The present invention sets up and has optimized the method that multiplex PCR-fluorescent mark single-basic extension-label microarray hybridization genotyping technique detects 6 gene label mononucleotide polymorphism sites such as people's complement receptor 1 type, so as to it at genetic characteristics of healthy people and study with the related of disease susceptibility.
Summary of the invention
The objective of the invention is to set up and adopt multiplex PCR-fluorescent mark single-basic extension-label microarray hybridization genotyping technique in conjunction with GenomeLab
TMSNPstream
The gene type system detects the method for people's complement receptor 1 type (CR1), Duffy antigen/Chemokine Receptors (DARC), leukocyte differentiation antigen 59 (CD59), leukocyte differentiation antigen 4 (CD4), leukocyte differentiation antigen 8A (CD8A) and leukocyte differentiation antigen 8B 6 gene label mononucleotide polymorphism sites such as (CD8B) simultaneously.
The method of the invention may further comprise the steps:
1. the screening of candidate gene tagged single-nucleotide polymorphic loci
Utilize the Tagger program of HapMap website to select the tagged single-nucleotide polymorphic loci (http://www.hapmap.org/cgi-perl/gbrowse/hapmap_B36/) of each gene: 1. to import the gene name; Condition: Population (crowd): CHB (China Han) 2. is set; Pairwise Methods (matching method): Tagger pairwise (Tagger match notation); RSquare cut off (multiple correlation coefficient threshold value): 0.8; MAF cut off (minimum gene frequency threshold value): 0.05; 3. carry out search, can show that this gene label mononucleotide polymorphism site reaches and the accession number of the mononucleotide polymorphism site of its close association.
The information that above-mentioned tagged single-nucleotide polymorphic loci is searched in login Pubmed website (http://www.ncbi.nlm.nih.gov/pubmed/).According to the allelic type of each mononucleotide polymorphism site, determine the type of the extension reagent that plan adopts, and adjust the mononucleotide polymorphism site do not meet design requirements in view of the above, mainly comprise: contain before and after the mononucleotide polymorphism site that a large amount of tumor-necrosis factor glycoproteinss, adjacent mononucleotide polymorphism site are too near in position on the karyomit(e), allelic gene type is not extending in the reagent sensing range.Final definite 27 tagged single-nucleotide polymorphic locis that detect see Table 1.
27 tagged single-nucleotide polymorphic loci information of table 1
2. mononucleotide polymorphism site amplimer and extension design of primers are with synthetic
The AP Editor software editing template sequence that utilizes Beckman company to write: in the accession number of the selected tagged single-nucleotide polymorphic loci of specified page input, be provided with and set up general class template (promptly the allelic gene type of all mononucleotide polymorphism sites replaces with T/A), executable operations can be searched for the sequence information that contains this site automatically; Software shields tumor-necrosis factor glycoproteins automatically with letter " N " simultaneously; The sequential file of deriving is the .txt form.
Utilize the online tool Autoprimer of Beckman company (http://www.autoprimer.com/) design mononucleotide polymorphism site amplimer and extend primer, selecting the design primer is that 48-is heavy, import the sequential file that AP Editor software is derived, carry out design procedure, can obtain the 1 pair of PCR primer of each mononucleotide polymorphism site and 1 and extend primer, extend 5 of primer ' end include with the microarray hybridization plate on fixed sequence program complementary base sequence.Adopt BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and UCSC In-Silico PCR instrument (http://genome.ucsc.edu/cgi-bin/hgPcr? command=start) specificity of PCR primer of design is estimated.The primer that designs entrusts Shanghai living worker's biotechnology company limited synthetic, adopts the polyacrylamide gel electrophoresis purifying.
27 mononucleotide polymorphism site amplimers of table 2 design and extend primer
3. the preparation of genomic dna sample
(1) people's Whole Blood Genomic DNA is extracted
Gather experimenter's empty stomach in early morning venous blood 2mL, go into the EDTA-2K anticoagulant tube, mixing.Adopt the Promega Wizard of company
Genomic dna purification kit (article No. A1125) extracts the human peripheral genomic dna, the by specification operation, concrete steps are as follows: 1. draw 900 μ L cell pyrolysis liquids and add in the 1.5mL Resins, epoxy pipe, the abundant mixing of jog blood specimen pipe, get whole blood 300 μ L and add in the Resins, epoxy pipe that contains cell pyrolysis liquid, put upside down mixed 5～6 times; 2. incubated at room 10min puts upside down 2～3 times therebetween and makes erythrocyte splitting, and centrifugal 40 seconds of 13000～16000r/min moves as far as possible and abandons supernatant, stays white depositions and about 10 μ L debris; About 10～15 seconds of concuss Resins, epoxy pipe makes white corpuscle resuspended; 3. add 300 μ L nucleus lysates to above-mentioned re-suspended cell, suction solution makes leukocytolysis 5～6 times; 4. add 1.5 μ LRNA enzymes, put upside down mixed 2～5 times, hatch 15min, be cooled to room temperature for 37 ℃; 5. add 100 μ L albumen precipitation liquid, concuss 10～20 seconds leaves standstill 10min, and concuss is 2～3 times therebetween, and albumen is fully precipitated; 6. put the centrifugal 5min of 13000～16000r/min in the room temperature, visible brown albumen precipitation moves to supernatant in the 1.5ml Resins, epoxy pipe that has added 400 μ L Virahols in advance; 7. put upside down mixed solution gently and become bulk until the thread DNA of white; 8. decant supernatant behind the centrifugal 1min of room temperature 13000～16000r/min, add 500 μ L, 70% ethanol, put upside down gently several times with washing DNA and Resins, epoxy tube wall, the centrifugal 1min of 13000～16000r/min; 9. decant supernatant, the Resins, epoxy pipe is inverted on the thieving paper, the air-dry 10～15min of room temperature; 10. after adding 4 ℃ of 100 μ L DNA hydrating fluids and spending the night, dna solution is put Ultralow Temperature Freezer preserve.
(2) DNA purity and concentration determination
1. DU800 ultraviolet spectrophotometer is transferred to the DNA tests state, instrument stabilizer 15min; 2. earlier with the air zeroing, draw 100 μ L sterilization bi-distilled water then to microcuvette, as blank, zeroing once more; 3. in 0.5mL Resins, epoxy pipe, add 96 μ L sterilization bi-distilled water, draw dna solution 4 μ L and add wherein, add to microcuvette behind the mixing, last machine test, record 260nm absorbancy/280nm absorbance ratio and concentration value.
4. substance PCR verifies mononucleotide polymorphism site amplimer quality
(1) prepares each substance PCR mixture respectively by following reaction system, add in 96 orifice plates.
Primer is to (every kind of primer concentration is 10 μ mol/L) 0.5 μ L
DNTPs (every kind of constituent concentration is 2.5mmol/L) 2.0 μ L
10 * PCR damping fluid II, 2.5 μ L
MgCl
2(25mmol/L) 5.0μL
Amplitaq Gold archaeal dna polymerase (5 units/μ L) 0.2 μ L
Genomic dna 0.5 μ L
Bi-distilled water 14.3 μ L
Add up to 25.0 μ L
(2) with shrouding film phonograph seal 96 orifice plates, centrifugal 10 seconds of 1000r/min.
(3) the following program of operation on gene-amplificative instrament: keep (HOLD): 94 ℃ of 1min → 40 circulations: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of 1min → HOLD:4 ℃ of 10min.
(4) 1.8% agarose gel electrophoresis: take by weighing the 0.9g agarose and add Erlenmeyer flask, measure 50mL 1 * Tris alkali-acetate-ethylenediamine tetraacetic acid (EDTA) damping fluid, shake up, put into microwave oven and be heated to agarose and dissolve fully; Take out postcooling to about 70 ℃, add the ethidium bromide solution that 2 μ L concentration are 10 μ g/ μ L, mixing is slowly poured agarose in the glue mould, inserts comb, treats that gelling is solid; In corresponding aperture, add sample and DL2000 dna molecular standard, glue is placed the electrophoresis chamber that is added with 1 * Tris alkali-acetate-ethylenediamine tetraacetic acid (EDTA) damping fluid, carry out electrophoresis with 80 volts of voltages; After electrophoresis finishes, adopt SX-300 gel images analytical system scanning gel, judge whether contain in the amplified production and target fragment fragment of the same size according to the dna molecular standard.
5. the detection of mononucleotide polymorphism site
(1) dilution of genome DNA sample
According to the DNA concentration of measuring, in 96 orifice plates, add an amount of sterilization bi-distilled water, DNA sample to be measured all is diluted to 10ng/ μ L.
(2) primer dilution
1. PCR primer dilution (totally 27 * 2):
Synthetic good primer is diluted to 240 μ mol/L; From the good PCR primer of 54 pipe dilutions, respectively get 5 μ L and join in the new Resins, epoxy pipe, and add 210 μ L sterilization bi-distilled water, as new multiple PCR primer pond; Have 480 μ L reagent in the PCR primer pond, each primer final concentration is 2.5 μ mol/L.
2. extend primer dilution (totally 27):
Synthetic good primer is diluted to 240 μ mol/L; From the good extension primer of 27 pipe dilutions, respectively get 5 μ L and join in the new Resins, epoxy pipe, and add 105 μ L sterilization bi-distilled water as new extension primer pond; The extension primer has 240 μ L reagent in the pond, and each primer final concentration is 5 μ mol/L.
(3) the heavy PCR of 27-
1. prepare the multiple reaction mixture, the required reagent of each 96 orifice plate (96 samples) is composed as follows:
Primer pond (each primer concentration is 2.5 μ mol/L) 10.56 μ L
DNTPs (each constituent concentration is 2.5mmol/L) 18.77 μ L
10 * PCR damping fluid II, 52.8 μ L
MgCl
2(25mmol/L) 105.6μL
Amplitaq Gold archaeal dna polymerase (5 units/μ L) 10.56 μ L
Bi-distilled water 118.51 μ L
Add up to 316.8 μ L
2. add the PCR mixture that 3 μ L prepare in the every hole of 96 orifice plates, will dilute good genomic dna again and join in the corresponding hole, every hole adds 2 μ L.
3. use shrouding film phonograph seal 96 orifice plates, centrifugal 10 seconds of 1000r/min.
4. operation following program: HOLD:94 ℃ 1min → 40 circulations on gene-amplificative instrament: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of 1min → HOLD:4 ℃ of 10min.
(4) PCR product purification
1. according to the system in the following table, prepare purified reagent:
Exonuclease I (20 units/μ L) 10.56 μ L
Alkaline phosphatase (1 unit/μ L) 105.13 μ L
10 * alkaline phosphatase damping fluid, 31.68 μ L
Bi-distilled water 169.43 μ L
Add up to 316.8 μ L
2. after PCR finishes, take out 96 orifice plates, centrifugal 10 seconds of 1000r/min, every hole adds the purified reagent of the new preparation of 3 μ L.
3. get new shrouding film phonograph seal 96 orifice plates, centrifugal 10 seconds of 1000r/min.
4. on gene-amplificative instrament, move following program: 37 ℃ of 30min → 96 ℃ 10min → 4 ℃ of 10min.The PCR product that purifying is good can be deposited several days under-20 ℃ of conditions.
(5) primer extension reaction
1. good PCR product thaws micro-array chip to be extended dilution buffer liquid and purifying in advance.
2. system shown in the according to the form below is prepared micro-array chip primer extension reaction mixture:
Extend dilution buffer liquid 397.17 μ L
Extend primer mixture 3.17 μ L
20 * extension reagent mixture (T/C, A/G) each 10.56 μ L
Bi-distilled water 315.53 μ L
Archaeal dna polymerase 2.21 μ L
Add up to 739.2 μ L
3. with centrifugal 10 seconds of PCR plate 1000r/min, in each hole, add the primer extension reaction mixture that 7 μ L prepare.
4. seal up the PCR plate with the shrouding film again, centrifugal 10 seconds of 1000r/min.
5. on gene-amplificative instrament, move following program: HOLD:96 ℃ 3min → 46 circulations: 94 ℃ 20 seconds, 40 ℃ of 11 seconds → HOLD:4 ℃ 10min.
(6) prepare microarray hybridization plate and carry out hybridization
1. according to the preparation of system shown in following table microarray hybridization plate washings:
20 * lavation buffer solution I, 300 μ L
Bi-distilled water 5700 μ L
Add up to 6000 μ L
2. get one 384 hole microarray hybridization plate, this obsolete hole is sealed with the shrouding film.In each reacting hole of hybridization plate, add 18 μ L, 1 * micro-array chip plate lavation buffer solution I.
3. the microarray hybridization plate being turned over is placed on the dust-free paper, is placed on the centrifugal 2min of 2000r/min in the pallet of whizzer together, makes that the scavenging solution in each hole all breaks away from the hybridization plate.
2. and 3. 4. repeating step cleans 3 times altogether.
5. according to the preparing hybrid of system shown in following table solution:
Hybridization buffer 850 μ L
Hybridizing reagent 50 μ L
Add up to 900 μ L
6. in the every hole of 96 orifice plates, add the above-mentioned hybridization solution of 8 μ L, centrifugal slightly on whizzer it is mixed.Drawing 18 μ L joins in the hole corresponding on the microarray hybridization plate.
The microarray hybridization plate that 7. will add mixed solution is put into the wet box of a sealing, places incubator, hatches 2～2.5 hours for 42 ℃.
(7) clean the hybridization plate behind the hybridization
1. according to the preparing hybrid of system shown in following table plate scavenging solution:
64 * lavation buffer solution II, 100 μ L
Bi-distilled water 6300 μ L
Add up to 6400 μ L
2. after hybridization is finished, will hybridize plate and be buckled on the dustless thieving paper, centrifugal 10 seconds of 2000r/min gets rid of reaction solution.
3. each reacting hole adds 18 μ L, 1 * lavation buffer solution II, cleans the microarray hybridization plate once more, cleans altogether 3 times.
4. after cleaning finishes, dip in the dehydrated alcohol wiping hybridization backboard face slide gently that takes a morsel, make its clear no vestige with dust-free paper.
(8) scanning hybridization plate
Cleaned hybridization plate is put into GenomeLab at once
TMSNPstream
On the gene type system scan instrument, operation RunManager program scans.Hybridization plate in scanning back keeps in Dark Place.
6. data analysis
(1) editor's sample and information
Operation PlateExplorer program, editor's sample information also generates the .txt file.The mononucleotide polymorphism site primer file that importing is derived from Autoprimer software is uploaded the sample information file, and all data are saved in the database.
(2) read scanning information
Operation SNPAdmin program is converted into graphic information with the hybridization plate information that scans, and uploads data, obtains genotype.
(3) the somatotype result adjusts and analyzes
Operation GetGenos program judges whether to exist offset point by QC Review, then need adjust as existing, and be saved to database, obtains gene type information then again.Analytical data under QC Review pattern, under the qualified situation of Quality Control point somatotype result, remove the mononucleotide polymorphism site of somatotype success ratio＜90%, derive each mononucleotide polymorphism site gene type report of somatotype success ratio＞90% by the Report program.
7. mononucleotide polymorphism site detects estimation of stability
To part dna sample mononucleotide polymorphism site replication 2 times, calculate single mononucleotide polymorphism site gene type coincidence rate as a result as stated above.
Description of drawings
Fig. 1 is Quality Control point somatotype result after the 91 routine pattern detection, and wherein a, b, c, d figure are respectively genotype XX, YY, XY and negative Quality Control point;
Fig. 2 is the somatotype result of 3 mononucleotide polymorphism sites after the 91 routine sample detection, wherein a, b, c figure are respectively mononucleotide polymorphism site rs3213427, rs863002, rs3020729, as seen 3 mononucleotide polymorphism sites of 91 routine sample evidences clearly are referred to XX, XY respectively, YY, XY, XX, XY, YY genotype.
Beneficial effect of the present invention:
(1) the Single base extension method of the present invention's employing makes the susceptibility of detection and accuracy substantially exceed general sequencing reaction;
(2) the label microarray hybridization technology of the present invention's employing can realize high flux, automation and Multiple detection;
(3) it is cheap that the method for the invention is simple to operate, the result judges quick and precisely, testing result stability is high, single detects average unit cost.
The applied reagent of the present invention is as follows: WizardGenomic DNA purification kit, agarose, alkaline phosphatase (1 unit/μ L), 10 * alkaline phosphatase buffer solution are U.S. Promega company product, dNTPs (every kind of constituent concentration is 2.5mmol/L), DL2000DNA molecular criteria are Japanese Takara company product, 10 * PCR buffer solution II, MgCl2(25mmol/L), Amplitaq Gold archaeal dna polymerase (5 units/μ L) is Switzerland Roche company product, Tris alkali-acetic acid-ethylenediamine tetra-acetic acid buffer solution, ethidium bromide solution (10 μ g/ μ L) is sky, Beijing root company product, exonuclease I (20 units/μ L) is U.S. Biolabs company product, extend dilution buffer liquid, 20 * extension reagent mixture (T/C, A/G), archaeal dna polymerase, 20 * lavation buffer solution I, hybridization buffer, hybridizing reagent, 64 * lavation buffer solution II, the microarray hybridization plate is U.S. Beckman Coulter company product (except indicating the source).
The applied key instrument of the present invention is as follows:
DU800 ultraviolet/visible light spectrophotometer, GenomeLabTM SNPstream
The Genotyping system is U.S. BeckmanCoulter company product, gene-amplificative instrament is German Eppendorf company product, the digital two permanent electrophoresis apparatuses of EPS-4105 are Beijing Jun Keyuan instrument plant product, and SX-300 gel image analysis system is Shanghai Xiao Yuan scientific ﹠ technical corporation product (except indicating the source).
Sequence table
＜110〉Chinese People's Liberation Army General Hospital
＜120〉a kind of tagged single-nucleotide polymorphic loci method that detects six immunity-related genes of human
<160>81
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs4844600-R-TC-U41.
<400>1
AATAACCAGG?GCGGCATT 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs4844600-R-TC-U41.
<400>2
AATGCCCCAG?AATGGCTT 18
<210>3
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs4844600-R-TC-U41 and extends primer.
<400>3
TACCTATGAC?CAGCAAGCAC?GATATGTCCC?AATGGGAAAC?TCAAA
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs2255301-TC-U39.
<400>4
GAAAGGCAAA?GGTGGAGG 18
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs2255301-TC-U39.
<400>5
TGCTGGGAGG?AGCGCTAA 18
<210>6
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs2255301-TC-U39 and extends primer.
<400>6
CACGACAAGA?CAACAGATAC?GGGGTAGAGG?GGGACAGCGG?CGACA 45
<210>7
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs11585-TC-U40.
<400>7
TACCTTAATT?TCTAGGTGGG?TGC 23
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs11585-TC-U40.
<400>8
CCTCCTCTCC?CAGAGGCT 18
<210>9
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs11585-TC-U40 and extends primer.
<400>9
AAGTACCACG?TCAACGTCAC?TTCATTGTAG?GTAAGGACAT?TTTCT 45
<210>10
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs831629-TC-U34.
<400>10
ATTTCTTAGT?TCTGCTATGG?ATGC 24
<210>11
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs831629-TC-U34.
<400>11
TAGTGATAGC?ATCAGGTGAG?AAGA 24
<210>12
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs831629-TC-U34 and extends primer.
<400>12
CACTAGTCAT?AACGCAGCCT?TACTTAGGCA?AGTTCAAAAC?CTCCC 45
<210>13
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs3020729-TC-U38.
<400>13
TAAAATGAAG?TGGTGAGCTT?AACC 24
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs3020729-TC-U38.
<400>14
GGCCCCTCTG?CAATGCAA 18
<210>15
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs3020729-TC-U38 and extends primer.
<400>15
ACGTAAGACC?ACTCAAGACC?AAAGAAAATC?TCTGTGAAAC?CCCTA 45
<210>16
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs1075835-TC-U37.
<400>16
TAGACTGTCA?TCTCTGTCCT?CAAG 24
<210>17
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs1075835-TC-U37.
<400>17
TACTCTGAAG?TCCCACAGCA?C 21
<210>18
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs1075835-TC-U37 and extends primer.
<400>18
ACCGCACTAA?GCAATGTATC?GGAGAGTTGA?GAAACCAAAT?CACTA 45
<210>19
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs13024609-R-TC-U35.
<400>19
TGGTTCTGAG?GCTACAGCA 19
<210>20
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs13024609-R-TC-U35.
<400>20
ACTTGAGTTA?AGATCACACT?GGG 23
<210>21
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs13024609-R-TC-U35 and extends primer.
<400>21
CAGAATAGCC?ACGCCTAGAT?ACCGAACTCG?GCCTGTGCCC?CAACT 45
<210>22
<211>28
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs863002-TC-U36.
<400>22
CTCAGTCTTT?ATATCTCTT?CCTTTTCC 28
<210>23
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs863002-TC-U36.
<400>23
ATGGAACTGA?CTCAAAGGCA 20
<210>24
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream extension primer of amplification rs863002-TC-U36.
<400>24
CACCGCTATC?AACAGACTTG?CTAGGAGGCT?AGCATAGGAA?GGAGA 45
<210>25
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs3213426-R-TC-U33.
<400>25
AACTTGTAGA?ATTACAACT?GGCAG 25
<210>26
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs3213426-R-TC-U33.
<400>26
TCCTCTGTGG?TCCCAGGG 18
<210>27
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs3213426-R-TC-U33 and extends primer.
<400>27
CGCAGAAGCA?ACTCACTTCT?GCCCAGGACC?ACAACCCACC?TCACT 45
<210>28
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs3213427-TC-U31.
<400>28
TTGAGTGTTG?CTCTCTAGTT?TCC 23
<210>29
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs3213427-TC-U31.
<400>29
CCTGGGATCC?AAATGAGC 18
<210>30
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs3213427-TC-U31 and extends primer.
<400>30
CAGAACATCC?TCAGAAGCAA?CCTTCAAGCC?TAGCCCTTCT?CTCAT 45
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs1055141-TC-U32.
<400>31
CAGAAAAATT?TGACCTGTGA?GG 22
<210>32
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs1055141-TC-U32.
<400>32
TTCTCCCGCT?TCGAGACC 18
<210>33
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs1055141-TC-U32 and extends primer.
<400>33
CAAGCAACGA?CCTACTACAA?CCACCTCCCC?TAAGCTGATG?CTGAG 45
<210>34
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs1075838-TC-U29.
<400>34
CCACTTTATA?TCCTAGGCCT?GG 22
<210>35
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs1075838-TC-U29.
<400>35
TAACTGGGGA?CCAGATGACA 20
<210>36
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs1075838-TC-U29 and extends primer.
<400>36
AATAAGCTCA?CCACCGTCAA?TCCTTTGAGG?TGCTATCTTG?GTGCC 45
<210>37
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs1546727-R-TC-U23.
<400>37
CTCTAGAGCT?GGCCTGT℃TA?TTT 23
<210>38
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs1546727-R-TC-U23.
<400>38
TAAACCTAGG?GTTCTCATTT?TCC 23
<210>39
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream extension primer of amplification rs1546727-R-TC-U23.
<400>39
AGTAGCCTAA?CAGCACTCGA?AGGTTAAGTA?ACTTGCCTGA?GGGTC 45
<210>40
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs13400210-TC-U25.
<400>40
ATAAATAAAA?AAGAGAAGCA?CAATGC 26
<210>41
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs13400210-TC-U25.
<400>41
AACCACCCTG?GAAGGTAGG 19
<210>42
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs13400210-TC-U25 and extends primer.
<400>42
CCATAACAAC?TTACCAGCCA?ATCTCTGACT?CTGGCCCGTC?TCTTC 45
<210>43
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs11064410-R-TC-U21.
<400>43
TCTCCTCAGT?TCTAGGCACA?C 21
<210>44
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs11064410-R-TC-U21.
<400>44
TTTTCTTTTG?AAGAGAAGCA?AGG 23
<210>45
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs11064410-R-TC-U21 and extends primer.
<400>45
GATCCATCAA?CAGACATCAC?TATCCTGCCA?TCACCCCAAA?CGCAA 45
<210>46
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs17048010-TC-U19.
<400>46
AAATTTCTGC?CGTAATTCTC?TACC 24
<210>47
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs17048010-TC-U19.
<400>47
TATGGAGTTT?CTGTTATAAC?TGCC 24
<210>48
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs17048010-TC-U19 and extends primer.
<400>48
CCACTCAACT?CCACGAATAC?TTCAAGGCTG?CTCCTTGTTC?TAATA 45
<210>49
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs4832054-R-TC-U20.
<400>49
AAAAGAAGTT?TCTCTCTCCT?CATG 24
<210>50
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs4832054-R-TC-U20.
<400>50
ATTTGTCATG?TAATCCACAT?GTTC 24
<210>51
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream extension primer of amplification rs4832054-R-TC-U20.
<400>51
ACAACTACCG?ACGACAAGAC?TGGAGTCTAT?TCATGCATCA?CTTCA 45
<210>52
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs3818361-TC-U18.
<400>52
AAAGGACAGT?TCCAGAGCAC?T 21
<210>53
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs3818361-TC-U18.
<400>53
TTAGTGAGAT?GTGGCTACTG?AACTAC 26
<210>54
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs3818361-TC-U18 and extends primer.
<400>54
AACATCCACG?CAACTCATAC?GTTAGATATG?GGGCAATTTC?CTTTG 45
<210>55
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs11064404-TC-U14.
<400>55
TACAAAATTG?GAACACCACC 20
<210>56
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs11064404-TC-U14.
<400>56
GCTTTGCCAT?TTTAGCCTC 19
<210>57
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs11064404-TC-U14 and extends primer.
<400>57
AACATACAGA?CGCACTCCTC?AATGCTGATG?AGGATGTGGA?GAAAC 45
<210>58
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs1738548-TC-U12.
<400>58
AATTACTTGT?CTGAAATGGC?AGG 23
<210>59
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs1738548-TC-U12.
<400>59
AAGAAGCAGA?AAAAAGTCAT?GATG 24
<210>60
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs1738548-TC-U12 and extends primer.
<400>60
CCAGATCCTC?ACCATGTAAG?ACCTCAATCT?GTAGTACATA?TCAAG 45
<210>61
<211>28
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs11118167-TC-U10.
<400>61
ATTTAATTGA?GTCAATACTG?GTTATGTG 28
<210>62
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs11118167-TC-U10.
<400>62
AAAAGATAAG?GCATGAGAAA?TGAA 24
<210>63
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs11118167-TC-U10 and extends primer.
<400>63
TCCAGAATAG?ACAACAGACG?CCAATATGTG?AATATTATTA?TCTTA 45
<210>64
<211>31
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs2231454-R-TC-U6.
<400>64
TATACTTACT?ACTGTGAAGT?AGGTCTCAAC?T 31
<210>65
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs2231454-R-TC-U6.
<400>65
TCCCTAAAGT?TTGAGAACCA?CT 22
<210>66
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs2231454-R-TC-U6 and extends primer.
<400>66
ACAACTCACG?CAAGTACCAT?CACCAACTGG?TGCTAAAGAC?TAGGA 45
<210>67
<211>32
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs12477946-R-TC-U7.
<400>67
ATATTTATTC?TATCTGGAGT?ATACATTTGT?GA 32
<210>68
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs12477946-R-TC-U7.
<400>68
AAGGATTCGA?TCATTAGTGC?TG 22
<210>69
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs12477946-R-TC-U7 and extends primer.
<400>69
TACAAGCACG?CACTAGACAT?GAGATAGAGT?TCAAGTTTTA?CGTTT 45
<210>70
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs6722860-TC-U5.
<400>70
TTTGTCTTAC?ATCCAAGTAC?TGAAGT 26
<210>71
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs6722860-TC-U5.
<400>71
ATCTGAGAAT?TCAGAAAGCA?GATC 24
<210>72
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs6722860-TC-U5 and extends primer.
<400>72
CAACAAGTAA?TCCGCAGACT?CCAGGCTGCT?ACAGGGCTTG?AAGGT 45
<210>73
<211>27
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs12576440-R-TC-U4.
<400>73
TTTTAGTCTA?GGGAGTAGTT?TTACCAA 27
<210>74
<211>31
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs12576440-R-TC-U4.
<400>74
CCCAAATATT?CTATAATTAA?CCAAGTATTA?C 31
<210>75
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs12576440-R-TC-U4 and extends primer.
<400>75
ATCTAACGCA?CCTACGACCT?GAACATTGTC?ATGTGTGAAT?TTTCC 45
<210>76
<211>28
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs10768024-TC-U3.
<400>76
AGATTAAGGA?GATTATTTTG?GATTATTC 28
<210>77
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs10768024-TC-U3.
<400>77
TCGGTCATGA?GTCTTTGACA 20
<210>78
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs10768024-TC-U3 and extends primer.
<400>78
CAGCCATCCA?TTCACTATCT?GGGCCCAATG?TAATCCCAGG?GGTCA 45
<210>79
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream amplimer of amplification rs9429945-TC-U2.
<400>79
AATTTCCACA?AACTTAGTGG?CTT 23
<210>80
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream amplimer of amplification rs9429945-TC-U2.
<400>80
AGCCTCCACA?AGGAACACA 19
<210>81
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream of amplification rs9429945-TC-U2 and extends primer.
<400>81
CTCAGACTAC?GAATCCACGT?TGTATTCTCT?TCCAGTTCTG?GAGGC 45
Claims (2)
1. tagged single-nucleotide polymorphic loci method that detects six immunity-related genes of human, these genes and tagged single-nucleotide polymorphic loci are: complement receptor 1 type gene: rs3818361, rs9429945, rs4844600, rs17048010, rs11118167; Duffy antigen/Chemokine Receptors gene: rs863002; Leukocyte differentiation antigen 59 genes: rs1546727, rs831629, rs10768024, rs2231454, rs11585, rs1738548, rs12576440; Leukocyte differentiation antigen 4 genes: rs11064410, rs2255301, rs1075835, rs1075838, rs11064404, rs3213427, rs1055141, rs3213426; Leukocyte differentiation antigen 8A gene: rs3020729; Leukocyte differentiation antigen 8B gene: rs4832054, rs12477946, rs13024609, rs6722860, rs13400210 is characterized in that detection method comprises the steps:
(1) selects the tagged single-nucleotide polymorphic loci of each gene, determine to extend types of agents, and the primary election mononucleotide polymorphism site is estimated, adjusted;
(2) design mononucleotide polymorphism site amplimer and extension primer, authorized company is synthetic;
(3) preparation people Whole Blood Genomic DNA sample;
(4) substance PCR checking mononucleotide polymorphism site amplimer quality;
(5) detect the sample that mononucleotide polymorphism site is used by multiplex PCR-fluorescent mark single-basic extension-label microarray hybridization prepared in reaction for the gene type system;
(6) adopt the gene type system to detect the sample mononucleotide polymorphism site;
(7) adopt gene type system software analysis mononucleotide polymorphism site somatotype effect, remove the mononucleotide polymorphism site of somatotype success ratio＜90%, and utilize the mononucleotide polymorphism site of somatotype success ratio＞90% to determine idiotype;
(8), calculate the somatotype coincidence rate, with the stability of evaluation method to a plurality of dna sample mononucleotide polymorphism site duplicate detection.
2. a kind of tagged single-nucleotide polymorphic loci method that detects six immunity-related genes of human according to claim 1,27 tagged single-nucleotide polymorphic loci amplimers that it is characterized in that designing and extend primer sequence as primer sequence as described in the sequence 1～81.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010100751 CN101851673B (en) | 2010-01-26 | 2010-01-26 | Method for detecting tagged single-nucleotide polymorphic loci of six immunity-related genes of human |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010100751 CN101851673B (en) | 2010-01-26 | 2010-01-26 | Method for detecting tagged single-nucleotide polymorphic loci of six immunity-related genes of human |
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| CN101851673B CN101851673B (en) | 2013-06-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484559A (en) * | 2013-10-17 | 2014-01-01 | 于惠 | Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene |
| CN104694657A (en) * | 2015-03-20 | 2015-06-10 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting human erythrocyte Duffy blood type genotyping |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MXPA04008477A (en) * | 2002-03-01 | 2005-10-26 | Ravgen Inc | Methods for detection of genetic disorders. |
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2010
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484559A (en) * | 2013-10-17 | 2014-01-01 | 于惠 | Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene |
| CN104694657A (en) * | 2015-03-20 | 2015-06-10 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting human erythrocyte Duffy blood type genotyping |
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| Publication number | Publication date |
|---|---|
| CN101851673B (en) | 2013-06-12 |
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