CN101851673B - 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 6 genes such as 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 (CD8B), belong to the Medical Molecular Biology field.
Background technology
Research shows, red corpuscle has immunity to be sticked, directly kills antigen foreign matter, regulate complement activity, strengthen the panimmunity functions such as NK cell function, binding chemotactic factor, the adjusting of participation B cellular immunization, in the natural immunity and acquired immunity, plays a significant role.There is the panimmunity related substances in erythrocyte surface or endochylema, wherein of greatest concern with the chemokine receptor function of the immunoadhering functions of complement receptor 1 type and Duffy antigen/Chemokine Receptors, 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 cells is the main cell of realizing the acquired immunity function, its surperficial leukocyte differentiation antigen 4 and leukocyte differentiation antigen 8 are combined with major histocompatibility complex class I I class and I class antigen respectively, can strengthen the degree that the T cell is combined with antigen presenting cell or target cell, in the signal transduction of T cell proliferation and differentiation, play an important role, the T cell of the T cell of leukocyte differentiation antigen 4 positives and leukocyte differentiation antigen 8 positives has respectively immunity and assists and immunosuppression and direct killing function.Change has occurred at various diseases in the level of above-mentioned immune molecule, closely relevant with disease generation, development and prognosis, but at present the gene of these molecules and the association study of disease genetic susceptibility is had no to report.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refer to single nucleotide diversity on karyomit(e) and the DNA sequence polymorphism that causes, it has that density is high, inheritance stability, be easy to the characteristics such as automated analysis, be considered to the third generation genetic marker after restriction fragment length polymorphism, microsatellite polymorphism, can be used for the 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.Developed at present more than 20 and planted method for detecting single nucleotide polymorphism, 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; The DNA chip technology of development had met the needs that high-throughput, automatization detect afterwards, take the DNA chip as detection platform, in conjunction with above-mentioned reaction principle, developed multiple method for detecting single nucleotide polymorphism, but there are the shortcomings such as false positive rate is high, flux is dumb in general mononucleotide polymorphism chip.(the 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 combined based on single base extension-label microarray hybridization, dexterously allele-specific primers sequence and sequence label that can sequence specific is combined on chip are integrated on the single-basic extension primer, the advantages such as multiple reaction of the high sensitive of single base extension and 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 again micro-sequencing, template mediation dyestuff terminator to mix analysis or single nucleotide polymorphism authenticate technology (single-nucleotide polymorphism-identification technology, SNP-IT), be development method for detecting single nucleotide polymorphism out on the basis of dideoxy sequencing method.Its principle is: at first design 1 pair of PCR primer for amplify the fragment that contains the purpose mononucleotide polymorphism site from genomic dna, design again 1 extension primer with mononucleotide polymorphism site upstream sequence to be measured complementation, 3 of this primer ' hold 1 base complementrity that last base 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, and due to the characteristic of ddNTP, when extending 1 base, when the ddNTP added and mononucleotide polymorphism site base complementrity, reaction stops; Extend by detection the base type that fluorescence types that base sends judges mononucleotide polymorphism site.The method is based on the order-checking principle, but, owing to only needing 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 pre-synthesis oligonucleotide strand (label) at the bottom of the plate of glass-chip plate, with, by hybridization in conjunction with getting on, then scanning and detect its fluorescent mark with fluorescently-labeled oligonucleotide chain of these label complementations.Introduce chip technology and contribute to realize that high-throughput, automatization detect, and adopt the label microarray to make Multiple detection become possibility, can utilize the label be fixed on 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 system of 12-weight/48-, the micro-array chip that this system adopts is 384 special orifice plates, it at the bottom of hole, is 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, pre-synthesis to 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 multiple PCR primer and the corresponding multiple extension primer of online design of primers instrument Autoprimer design amplification containing candidate's mononucleotide polymorphism site purpose fragment, every extend 5 of primer ' end be added with chip on the sequence of sequence label complementation, for the combination of extension products and label microarray, result can be read by the Two Colour Fluorescence on 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, easy and simple to handle, and level of automation is high.
The present invention sets up and has optimized the method for multiplex PCR-fluorescent mark single-basic extension-6 gene label mononucleotide polymorphism sites such as label microarray hybridization genotyping technique detection people complement receptor 1 type, in order to its genetic characteristics at Healthy People is reached and studied with the associated 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 6 gene label mononucleotide polymorphism sites such as 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 (CD8B) simultaneously.
The method of the invention comprises the following 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 input 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 of intending employing, and adjust accordingly the mononucleotide polymorphism site do not meet design requirements, mainly comprise: too near containing a large amount of tumor-necrosis factor glycoproteinss, adjacent mononucleotide polymorphism site position on karyomit(e) before and after mononucleotide polymorphism site, allelic gene type is not in extending the reagent sensing range.Final definite 27 tagged single-nucleotide polymorphic locis that detect are in 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, arrange and set up general class template (the allelic gene type of all mononucleotide polymorphism sites replaces with T/A), executable operations, get final product the sequence information of automatic search containing this site; Software is automatically by letter " N " shielding for tumor-necrosis factor glycoproteins 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 the base sequence of fixed sequence program complementation.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 designed entrusts Shanghai living work 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 limosis vein blood in early morning 2mL, enter the EDTA-2K anticoagulant tube, mix.Adopt the Wizard of Promega 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 1.5mL epoxy resin pipe, jog blood specimen pipe fully mixes, get whole blood 300 μ L and add in the epoxy resin pipe that contains cell pyrolysis liquid, put upside down mixed 5～6 times; 2. incubated at room 10min, put upside down 2～3 times therebetween and make erythrocyte splitting, and centrifugal 40 seconds of 13000～16000r/min, move as far as possible and abandon supernatant, stays white depositions and about 10 μ L debris; Concuss epoxy resin pipe approximately 10～15 seconds, make 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, standing 10min, concuss is 2～3 times therebetween, and albumen is fully precipitated; 6. put the centrifugal 5min of 13000～16000r/min in room temperature, visible brown albumen precipitation, move to supernatant in the 1.5ml epoxy resin pipe that has added in advance 400 μ L Virahols; 7. put upside down mixed solution gently until white thread DNA becomes bulk; 8. decant supernatant after 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 epoxy resin tube wall, the centrifugal 1min of 13000～16000r/min; 9. decant supernatant, the epoxy resin pipe is inverted on 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 to Ultralow Temperature Freezer and preserve.
(2) DNA purity and concentration determination
1. DU800 ultraviolet/visible light spectrophotometer is adjusted to the DNA tests state, instrument stabilizer 15min; 2. first with the air zeroing, then draw 100 μ L sterilizing bi-distilled waters to microcuvette, as blank, zeroing again; 3. add 96 μ L sterilizing bi-distilled waters in 0.5mL epoxy resin pipe, draw DNA solution 4 μ L and add wherein, after mixing, add to microcuvette, examination with computer, record 260nm absorbancy/280nm absorbance ratio and concentration value.
4. substance PCR verifies mononucleotide polymorphism site amplimer quality
(1) prepare respectively each substance PCR mixture by following reaction system, add in 96 orifice plates.
Primer pair (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 the 0.9g agarose and add Erlenmeyer flask, measure 50mL 1 * Tris alkali-acetic acid-ethylenediamine tetraacetic acid (EDTA) damping fluid, shake up, put into microwave oven and be heated to agarose and dissolve fully; Be cooled to approximately 70 ℃ after taking-up, add the ethidium bromide solution that 2 μ L concentration are 10 μ g/ μ L, mix, agarose is slowly poured in the glue mould, insert comb, treat that gelling is solid; Add sample and DL2000 DNA molecular standard in corresponding aperture, glue is placed in to the electrophoresis chamber that is added with 1 * Tris alkali-acetic acid-ethylenediamine tetraacetic acid (EDTA) damping fluid, with 80 volts of voltages, carry out electrophoresis; After electrophoresis finishes, adopt SX-300 gel image analysis system scan gel, according to the DNA molecular standard, judge in amplified production whether contain and target fragment fragment of the same size.
5. the detection of mononucleotide polymorphism site
(1) dilution of genome DNA sample
DNA concentration according to measuring adds appropriate sterilizing bi-distilled water in 96 orifice plates, and DNA sample to be measured all is diluted to 10ng/ μ L.
(2) primer dilution
1. PCR primer dilution (totally 27 * 2):
Synthetic primer is diluted to 240 μ mol/L; Respectively get 5 μ L the PCR primer diluted from 54 pipes and join in a new epoxy resin pipe, and add 210 μ L sterilizing bi-distilled waters, as new multiple PCR primer pond; Have 480 μ L reagent in PCR primer pond, each primer final concentration is 2.5 μ mol/L.
2. extend primer dilution (totally 27):
Synthetic primer is diluted to 240 μ mol/L; Respectively get 5 μ L the extension primer diluted from 27 pipes and join in a new epoxy resin pipe, and add 105 μ L sterilizing bi-distilled waters as new extension primer pond; Extend in the primer pond and have 240 μ L reagent, 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. to the PCR mixture that adds 3 μ L to prepare in the 96 every holes of orifice plate, then the genomic dna diluted is joined to corresponding Nei，Mei hole, hole add 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 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. move following program on gene-amplificative instrament: 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 to dilution buffer liquid and purifying in advance.
2. system shown 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
Each 10.56 μ L of 20 * extension reagent mixture (T/C, A/G)
Bi-distilled water 315.53 μ L
Archaeal dna polymerase 2.21 μ L
Add up to 739.2 μ L
3. by centrifugal 10 seconds of PCR plate 1000r/min, to the primer extension reaction mixture that adds 7 μ L to prepare in each hole.
4. again with the shrouding film, seal up the PCR plate, 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 the 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 384 hole microarray hybridization plates, with the shrouding film, this obsolete hole is sealed.Add 18 μ L 1 * micro-array chip plate lavation buffer solution I in each reacting hole of hybridization plate.
3. the microarray hybridization plate is turned over and is placed on dust-free paper, be placed on together the interior centrifugal 2min of 2000r/min of pallet of whizzer, make to hybridize the scavenging solution in each hole on plate and all break away from.
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. add the above-mentioned hybridization solution of 8 μ L in the 96 every holes of orifice plate, centrifugal slightly on whizzer it is mixed.Drawing 18 μ L joins in hole corresponding on the microarray hybridization plate.
7. will add the microarray hybridization plate of mixed solution to put into the wet box of a sealing, be placed in incubator, hatch 2～2.5 hours for 42 ℃.
(7) clean the hybridization plate after 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 completes, will hybridize plate and be buckled on dustless thieving paper, centrifugal 10 seconds of 2000r/min, get rid of reaction solution.
3. each reacting hole adds 18 μ L 1 * lavation buffer solution II, again cleans the microarray hybridization plate, cleans altogether 3 times.
4. after cleaning, with dust-free paper, dip a small amount of dehydrated alcohol wiping hybridization backboard face slide gently, make it clear without vestige.
(8) scanning hybridization plate
Cleaned hybridization plate is put into GenomeLab at once
tMsNPstream
on gene type system scan instrument, operation RunManager program, scanned.After scanning, the hybridization plate keeps in Dark Place.
6. data analysis
(1) editing sample and information
Operation PlateExplorer program, editing sample information also generates the .txt file.The mononucleotide polymorphism site primer file that importing is derived from Autoprimer software, upload the sample information file, and all data are saved in database.
(2) read scanning information
Operation SNPAdmin program, be converted into graphic information by the hybridization plate information of scanning, and uploading data, obtains genotype.
(3) the somatotype result is adjusted and is analyzed
Operation GetGenos program, judge whether to exist offset point by QC Review, needs to adjust as existed, and be saved to database, then again obtains gene type information.Analytical data under QC Review pattern, in the situation that Quality Control point somatotype result is qualified, 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.
The accompanying drawing explanation
Fig. 1 is Quality Control point somatotype result after 91 routine pattern detection, and wherein a, b, c, d figure are respectively genotype XX, YY, XY and negative Quality Control point;
The somatotype result that Fig. 2 is 3 mononucleotide polymorphism sites after 91 routine sample detection, wherein a, b, c figure are respectively mononucleotide polymorphism site rs3213427, rs863002, rs3020729,3 mononucleotide polymorphism sites of visible 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-basic extension method that the present invention adopts makes the susceptibility and the accuracy that detect substantially exceed general sequencing reaction;
(2) the label microarray hybridization technology that the present invention adopts can realize high-throughput, automatization and Multiple detection;
(3) it is cheap that the method for the invention is simple to operate, result judges quick and precisely, detected result stability is high, single detects average cost.
The applied reagent of the present invention is as follows: Wizard
genomic dna purification kit, agarose, alkaline phosphatase (1 unit/μ L), 10 * alkaline phosphatase damping fluid 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 damping fluid II, MgCl
2(25mmol/L), Amplitaq Gold archaeal dna polymerase (5 units/μ L) is Switzerland Roche company product, Tris alkali-acetic acid-ethylenediamine tetraacetic acid (EDTA) damping fluid, ethidium bromide solution (10 μ g/ μ L) is Beijing Tian Gen 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 source).
The applied key instrument of the present invention is as follows:
DU800 ultraviolet/visible light spectrophotometer, GenomeLab
tMsNPstream
the gene type 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 little Yuan scientific & technical corporation product (except indicating 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 (1)
1. a 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) select the tagged single-nucleotide polymorphic loci of each gene, determine and extend types of agents, and the primary election mononucleotide polymorphism site is estimated, adjusted;
(2) design and synthesize 27 tagged single-nucleotide polymorphic loci amplimers and extend primer sequence, above as primer sequence as described in sequence 1～81;
(3) prepare people's Whole Blood Genomic DNA sample;
(4) substance PCR checking mononucleotide polymorphism site amplimer quality;
(5) detect the sample of mononucleotide polymorphism site for the gene type system by multiplex PCR-fluorescent mark single-basic extension-label microarray hybridization reaction preparation;
(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 somatotype success ratio 90% mononucleotide polymorphism site determines idiotype;
(8), to a plurality of DNA sample mononucleotide polymorphism site duplicate detection, calculate the somatotype coincidence rate, with the stability of evaluation method.
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| 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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| CN103484559B (en) * | 2013-10-17 | 2015-07-15 | 李彩香 | 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 | Priority date | Publication date | Assignee | Title |
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| CN1650031A (en) * | 2002-03-01 | 2005-08-03 | 拉瓦格恩公司 | Rapid analysis of variations in a genome |
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| CN1650031A (en) * | 2002-03-01 | 2005-08-03 | 拉瓦格恩公司 | Rapid analysis of variations in a genome |
Non-Patent Citations (2)
| Title |
|---|
| 张阳东等.《2型糖尿病患者C反应蛋白及rs1205多态性研究》.《标记免疫分析与临床》.2009,第16卷(第1期),全文. * |
| 李立青等.《标签微阵列技术高通量测定单核苷酸多态性方法的建立与应用》.《解放军医学杂志》.2009,第34卷(第2期),摘要、材料与方法、结果. * |
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