CN108531602A - A kind of primer and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility - Google Patents
A kind of primer and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility Download PDFInfo
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Abstract
The present invention provides a kind of primers and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility, which includes the primer in the sites rs872071, the primer in the sites rs2647012, the primer in the sites rs1045241, the primer in the sites rs6773854 and the primer in the sites rs6421571.Using the method for the relevant SNP site polymorphism of primer detection lymph cancer neurological susceptibility, include the following steps：(1) it acquires sample and extracts DNA；(2) PCR amplification of target gene, glue recycling are carried out using above-mentioned primer respectively；(3) concentration for measuring glue recovery product, carries out fluorescent marker PCR reactions, and purify to reaction product；(4) machine in purified product is sequenced, and SNP testing results is analyzed.The primer specificity is good, high sensitivity, accuracy are good, and detection method is simple, and predictable subject suffers from the risk of lymph cancer.
Description
Technical field
The invention belongs to biotechnologies, and in particular to one kind is for detecting the relevant SNP site of lymph cancer neurological susceptibility
Primer and detection method.
Background technology
Lymph cancer is also known as lymthoma, is to be primary in lymph node or the malignant tumour of other lymphoid tissues, is that China is common
One of ten big malignant tumours.The disease be more common in, it is young, male patient is more than women, can be divided by the difference of its cell component
Hodgkin lymphoma and non-Hodgkin lymphoma two major classes, molecular genetics cause lymphocyte differentiation developmental disorder to be leaching extremely
The important mechanisms of bar carcinogenesis, complicated biological behaviour so that clinical treatment is extremely difficult.
Lymphatic system is that human body infects " main force " to Anti-bacterium, is human immunity " natural cover for defense ".But due to existing
For the aggravation of social competition, the accelerating rhythm of life, operating pressure, the psychological pressure of people is also increasing, and environment is dirty in addition
The getting worse of dye makes people also be greatly increased by the chance of virus and bacterium infection.In addition, not due to people some of itself
Good life-form structure and eating habit so that the function of lymphatic system defence germ weakens significantly, and the incidence of lymph cancer is in year by year
Growth trend.
Currently, traditional medicine is widely used to the inspection and diagnosis of lymph cancer, but inspection result often has suffered from lymph
Cancer, a large amount of clinical tests confirm that 60%-70% early stages patients with non Hodgkin lymphoma can be cured using immunochemotherapy, still
If cannot timely diagnosis and treatment, patient vitals will be seized in possible half a year or 1-2.So lymph cancer it is more early discovery and it is timely
It is bigger to cure chance for diagnosis and treatment.
In conclusion by detecting the polymorphism with the relevant SNP site of lymph cancer neurological susceptibility, can not only be lost from molecule
It passes and understands cell carcinogenesis mechanism in level, and be of great significance to the prediction, prevention, genetic counselling etc. of lymph cancer.
Invention content
For the above-mentioned problems in the prior art, the present invention provides a kind of relevant for detecting lymph cancer neurological susceptibility
The primer and detection method of SNP site, primer specificity is good, and detection method accuracy is high, and the risk that can be used for lymph cancer is commented
Estimate, guidance is provided for the disease prevention of lymph cancer.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of primer for detecting the relevant SNP site of lymph cancer neurological susceptibility, these primers include the sites rs872071
Primer, the primer in the sites rs2647012, the primer in the sites rs1045241, the sites rs6773854 primer and
The primer in the sites rs6421571；
Wherein, the primer in the sites rs872071 is F：5′-GAACAACTCTGGGAGTCT-3′(SEQ ID No：And R 1)：
5′-CTCTCTGAAGGAACGTAAC-3′(SEQ ID No：2)；
The primer in the sites rs2647012 is F：5′-GATATGCAGTATTAGGTGGT-3′(SEQ ID No：And R 3)：5′-
TAGACCAGTCAGTTGCTTAC-3′(SEQ ID No：4)；
The primer in the sites rs1045241 is F：5′-GTGGATTATACCTTTGACC-3′(SEQ ID No：And R 5)：5′-
GTCGATGACTCAATCTTAAC-3′(SEQ ID No：6)；
The primer in the sites rs6773854 is F：5′-CTTTACCAATCCACAGACTA-3′(SEQ ID No：And R 7)：5′-
GCTGTGTAATGATGAAGAC-3′(SEQ ID No：8)；
The primer in the sites rs6421571 is F：5′-CTTTCACTGCTGAGTTAGTT-3′(SEQ ID No：And R 9)：5′-
CTCACCAGTTTATCGTTAGT-3′(SEQ ID No：10).
Using the method for the relevant SNP site polymorphism of above-mentioned primer detection lymph cancer neurological susceptibility, include the following steps：
(1) it acquires sample and extracts its genomic DNA；
(2) it carries out the PCR amplification of target gene to genomic DNA respectively using above-mentioned primer, and amplified production is carried out
Glue recycles；
(3) concentration mensuration is carried out to glue recovery product, then calculation template amount carries out PCR amplification (fluorescent marker reaction)
And it purifies；
(4) by the purified product in step (3) in the full-automatic sequenator of 3730 types (U.S. Applied
Biosystems companies) loading, SNP genotype is analyzed with Chromas softwares.
Further, PCR amplification system is in step (2)：DNA profiling content is 100-150ng, a concentration of 5pmol/ μ L
Forward primer 3.0 μ L, a concentration of 5pmol/ μ L 3.0 μ L, Prime STAR MAX of reverse primer, 25.0 μ L, use ddH2O is mended
Sufficient volume is to 50 μ L.
Further, pcr amplification reaction condition is in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 15s, 72 DEG C of extension 15s carry out 30 cycles, last 72 DEG C of extensions 5min.
Further, PCR amplification system is in step (3)：DTCS Master Mix 2.5 μ L, a concentration of 5pmol/ μ L
Reverse primer 1.0 μ L, DNA profiling 20ng use ddH2O supplies volume to 10 μ L.
Further, pcr amplification reaction condition is in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C are moved back
Fiery 25s, 60 DEG C of extension 3min, 30 cycles, last 60 DEG C of extensions 20min.
Provided by the present invention for detect the relevant SNP site of lymph cancer neurological susceptibility primer and detection method, have with
Lower advantageous effect：
(1) the present invention provides the primer for detecting lymph cancer susceptibility gene related locus, which has specificity
Advantage high, accuracy is good, realizes the detection of the relevant SNP site of lymph cancer neurological susceptibility, improves detection efficiency.
(2) the present invention also provides a kind of method of the detection relevant SNP site of lymph cancer neurological susceptibility, this method is direct
The advantages that PCR sequencing PCR, this detection method is simple, and testing result also has specific good, high sensitivity, and accuracy is good, Ke Yiwei
The disease prevention of lymph cancer provides guidance.
Description of the drawings
Fig. 1 is the agarose that different subject's blood DNA samples carry out PCR amplification products therefrom under 5 kinds of different primers
Gel electrophoresis figure；
Fig. 2 is the sequencing result figure of rs872071 loci polymorphisms detection；
Fig. 3 is the sequencing result figure of rs2647012 loci polymorphisms detection；
Fig. 4 is the sequencing result figure of rs1045241 loci polymorphisms detection；
Fig. 5 is the sequencing result figure of rs6773854 loci polymorphisms detection；
Fig. 6 is the sequencing result figure of rs6421571 loci polymorphisms detection；
Fig. 7 is the fluorescence proof diagram of rs872071 loci polymorphism testing results；
Fig. 8 is the fluorescence proof diagram of rs2647012 loci polymorphism testing results；
Fig. 9 is the fluorescence proof diagram of rs1045241 loci polymorphism testing results；
Figure 10 is the fluorescence proof diagram of rs6773854 loci polymorphism testing results；
Figure 11 is the fluorescence proof diagram of rs6421571 loci polymorphism testing results.
Specific implementation mode
The primer of the design amplification SNP site of embodiment 1
A large amount of primer is devised for the relevant SNP site of lymph cancer neurological susceptibility, passes through the optimization of primer reaction condition
With compare, filter out the good five pairs of primers of specificity, including：Positioned at the sites rs872071 of IRF4 genes primer, be located at
The primer in the sites rs2647012 of HLA-DQB1 genes, positioned at the sites rs1045241 of TNFAIP8 genes primer, be located at
The primer in the sites rs6773854 of BCL6 genes and primer positioned at the sites rs6421571 of CXCR5 genes.
Wherein, the primer in the sites rs872071 is F：5′-GAACAACTCTGGGAGTCT-3′(SEQ IDNo：And R 1)：
5′-CTCTCTGAAGGAACGTAAC-3′(SEQ ID No：2)；
The primer in the sites rs2647012 is F：5′-GATATGCAGTATTAGGTGGT-3′(SEQ IDNo：And R 3)：5′-
TAGACCAGTCAGTTGCTTAC-3′(SEQ ID No：4)；
The primer in the sites rs1045241 is F：5′-GTGGATTATACCTTTGACC-3′(SEQ ID No：And R 5)：5′-
GTCGATGACTCAATCTTAAC-3′(SEQ ID No：6)；
The primer in the sites rs6773854 is F：5′-CTTTACCAATCCACAGACTA-3′(SEQ ID No：And R 7)：5′-
GCTGTGTAATGATGAAGAC-3′(SEQ ID No：8)；
The primer in the sites rs6421571 is F：5′-CTTTCACTGCTGAGTTAGTT-3′(SEQ ID No：And R 9)：5′-
CTCACCAGTTTATCGTTAGT-3′(SEQ ID No：10).
The rs codes sequence such as SEQ ID No of rs872071：Shown in 11, the rs codes sequence such as SEQ ID No of rs2647012：
Shown in 12, the rs codes sequence such as SEQ ID No of rs1045241：Shown in 13, the rs codes sequence such as SEQ ID No of rs6773854：
Shown in 14, the rs codes sequence such as SEQ ID No of rs6421571：Shown in 15.
PCR amplification, PCR are carried out respectively using the primer pair testing gene group DNA of the relevant SNP site of lymph cancer neurological susceptibility
Amplification system is：2 μ L of DNA profiling make its content for 100-150ng, 17 μ L of deionized water, and the forward direction of a concentration of 5pmol/ μ L is drawn
3.0 μ L, Primer STAR MAX of reverse primer, the 25.0 μ L of object 3.0 μ L, a concentration of 5pmol/ μ L；Amplification reaction condition is：
95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 15s, 72 DEG C of extension 15s, 30 cycles, last 72 DEG C extend 5min
Afterwards in 4 DEG C of preservations；By amplified production into row agarose gel electrophoresis, the results are shown in Figure 1, wherein 1 hole is the sites rs872071
Primer amplification band；2 holes are the primer amplification band in the sites rs2647012；3 holes are the primer amplification in the sites rs1045241
Band；4 holes are the primer amplification band in the sites rs6773854；5 holes are the primer amplification band in the sites rs6421571.
As shown in Figure 1, purpose band is clear, no miscellaneous band, and clip size and design is in the same size, illustrates drawing for design
Object specificity is good.
The detection of embodiment 2 and the relevant SNP site polymorphism of lymph cancer neurological susceptibility
(1) extraction of genomic DNA
Subject peripheral blood 5mL is acquired with EDTA anticoagulant tubes, the extracting method of genomic DNA is with reference to poba gene group
The specification of DNA extraction kit (be purchased from Beijing Tiangeng biochemical technology Co., Ltd), the operating procedure in by specification carry out
Extraction.The complete genome DNA of acquisition using ultramicron nucleic acid-protein analyzer (cypress essence BioDrop μ Lite) detect its concentration with
And purity, record initial data.The DNA that concentration and purity are all reached to requirement is placed in -20 DEG C of refrigerators and saves backup.
(2) PCR amplification of target gene, electrophoresis and glue recycling
PCR amplification：With the above-mentioned site rs872071, rs2647012, rs1045241, rs6773854, rs6421571
Primer carries out PCR amplification to the DNA of extraction respectively.Reaction system is 50 μ L, specific as shown in table 1, amplification program such as 2 institute of table
Show.After the completion of PCR reactions, takes out 4 DEG C of PCR product and preserve and (- 20 DEG C of placement is needed to freeze overnight).
1 PCR reaction systems of table
| Reagent | Volume (μ L) |
| DNA | 2(100-150ng) |
| ddH2O | 17 |
| Primer F | 3.0 |
| Primer R | 3.0 |
| Primer STAR MAX | 25.0 |
| total | 50.0 |
2 PCR amplification program of table
After PCR amplification, 50 μ L amplified productions are taken, using 2% agarose gel electrophoresis, electrophoretic parameters are voltage
120V, electric current 400mA, time 30min, specific band nothing but in electrophoresis result；In gel electrophoresis images point after the completion of electrophoresis
The gel containing target fragment is cut under analyzer, gel piece is fitted into EP pipes, and the method for glue recycling is with reference to TaKaRa
The specification of MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 kits, finally to glue recycling
Product carries out concentration mensuration, and 4 DEG C save backup.
(3) it marks, purify and is sequenced
Label：A clean 0.2mL PCR pipe is taken, is sequentially added：DTCS Master Mix, sequencing primer, sterilizing are super
The dosage of pure water, template DNA, wherein template DNA and sterilizing ultra-pure water is determined by the DNA concentration that previous step is measured, and is added
Template DNA amount be 20ng, reaction system is 10 μ L, and specific then to carry out fluorescent marker PCR as shown in table 3, amplification program is shown in
Table 4；
3 PCR reaction systems of table
| Reagent | Volume (μ L) |
| DTCS Master Mix | 2.5 |
| Primer R | 1.0 |
| ddH2O | 6.5-b |
| Template DNA | b(20ng) |
| It amounts to | 10.0 |
4 PCR amplification program of table
Purifying：By a concentration of 3mol/L, sodium-acetate buffer, 0.1mol/L of the pH value for 5.2, pH value is 8.0
Na2Edta buffer liquid and Glycogen are with volume ratio for 2:2:1 prepares terminate liquid, then takes 5 μ L terminate liquids that above-mentioned fluorescence is added
It is mixed well in label reaction PCR product, centrifuges, liquid is transferred in a new 1.5mL EP pipe, 50 μ L are then added
Absolute ethyl alcohol is pre-chilled, mixes well, is put into after -20 DEG C of freezing 10min of refrigerator and centrifuges 5min in 12000r/min, discard supernatant,
150 μ L are added into EP pipes again, 70% ethyl alcohol is pre-chilled, be put into supercentrifuge centrifugation, 12000r/min centrifuges 2min, discards
Clearly, it slightly centrifuging, blots remaining liquid in EP pipes with pipettor, open EP pipe lids, room temperature is dried to white precipitate bleach, then
25 μ L SLS (Sample Loading Solution) dissolving DNA is added into EP pipes, stands 8min, brief centrifugation；
Sequencing：The DNA of dissolving is added in 96 hole sample plate holes, bubble cannot be generated during addition, is then added again
Enter appropriate mineral oil；One piece of 96 new orifice plate is taken, 10 drop dissociating buffers are added in corresponding aperture, ultra-pure water is added into glue groove
To liquid close to index line, it is (beautiful that 96 hole sample panels, 96 hole buffer solutions and glue groove are finally packed into 3730 full-automatic sequenators
Applied Biosystems companies of state) it is sequenced.
By sequence analysis software Chromas, sequencing result is compared with standard sequence, finds SNP site, pass through
Analyze the type of base at SNP site, so that it may to obtain the genotype of SNP site.
2-1,2-2 and 2-3 are the sequencing result figure of rs872071 loci polymorphisms detection in Fig. 2, and genotype is followed successively by
AA、AG、GG；Wherein AA is the non-susceptible genotype in the sites rs872071, and the tumor susceptibility gene that GG and AG is the sites rs872071
Type.
3-1,3-2 and 3-3 are the sequencing result figure of rs2647012 loci polymorphisms detection in Fig. 3, and genotype is followed successively by
GG、AG、AA；Wherein GG is the non-susceptible genotype in the sites rs2647012, and AG and AA is the easy of the sites rs2647012 site
Feel genotype.
4-1,4-2 and 4-3 are the sequencing result figure of rs1045241 loci polymorphisms detection in Fig. 4, and genotype is followed successively by
CC、CT、TT；Wherein CC is the non-susceptible genotype in the sites rs1045241, and the easy sensillary base that TT and CT is the sites rs1045241
Because of type.
5-1,5-2 and 5-3 are the sequencing result figure of rs6773854 loci polymorphisms detection in Fig. 5, and genotype is followed successively by
TT、TC、CC；Wherein TT is the non-susceptible genotype in the sites rs6773854, and the easy sensillary base that TC and CC is the sites rs6773854
Because of type.
6-1,6-2,6-3 are the testing result figure of the sites rs6421571 fluorescence probe method in Fig. 6, and genotype is followed successively by
CC、TC、TT；Wherein CC is the non-susceptible genotype in the sites rs6421571, and the easy sensillary base that TC and TT is the sites rs6421571
Because of type.
Above-mentioned sequencing result figure without set peak, background peaks, miscellaneous peak, float peak phenomena such as, illustrate using PCR sequencing PCR detection SNP
Point polymorphism, specificity is good, accuracy is high.Therefore, testing agency can according to the polymorphism of above-mentioned SNP site, assess by
Inspection person suffers from the risk of lymph cancer, and guidance is provided for the disease prevention of lymph cancer.
The verification of embodiment 3 and the relevant SNP site polymorphic detection result of lymph cancer neurological susceptibility
Above-mentioned testing result is verified using fluorescence probe method, result is as shown in Fig. 7-Figure 11.
Wherein, in Fig. 7 7-1,7-2 and 7-3 be rs872071 loci polymorphism testing results fluorescence proof diagram, gene
Type is followed successively by AA, AG, GG；Wherein AA is the non-susceptible genotype in the sites rs872071, and GG and AG is the sites rs872071
Susceptible genotype.
8-1,8-2 and 8-3 are the fluorescence proof diagram of rs2647012 loci polymorphism testing results in Fig. 8, genotype according to
Secondary is GG, GA, AA；Wherein GG is the non-susceptible genotype in the sites rs2647012, and GA and AA is the sites rs2647012 site
Susceptible genotype.
9-1,9-2 and 9-3 are the fluorescence proof diagram of rs1045241 loci polymorphism testing results in Fig. 9, genotype according to
Secondary is CC, CT, TT；Wherein CC is the non-susceptible genotype in the sites rs1045241, and TT and CT is the easy of the sites rs1045241
Feel genotype.
10-1,10-2 and 10-3 are the fluorescence proof diagram of rs6773854 loci polymorphism testing results, gene in Figure 10
Type is followed successively by TT, TC, CC；Wherein TT is the non-susceptible genotype in the sites rs6773854, and TC and CC is the sites rs6773854
Susceptible genotype.
11-1,11-2,11-3 are the fluorescence proof diagram of rs6421571 loci polymorphism testing results, gene in Figure 11
Type is followed successively by CC, TC, TT；Wherein CC is the non-susceptible genotype in the sites rs6421571, and TC and TT is the sites rs6421571
Susceptible genotype.
Above-mentioned fluorescence probe method testing result is consistent with PCR sequencing PCR testing result 100%, further demonstrates using sequencing
Method detects the specificity and accuracy of SNP site polymorphism.
Sequence table
<110>Co., Ltd of medical test institute of Chengdu Zhong Chuanqing sections
<120>A kind of primer and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility
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ttatttgtat tacgtatctc ccgaagtcct ctgtggccag ctgcatctgt ctgaatggtg 300
cgtgaaggct ctcagacctt acacaccatt ttgtaagtta tgttttacat gccccgtttt 360
tgagactgat ctcgatgcag gtggatctcc ttgagatcct gatagcctgt tacaggaatg 420
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atttagctcc tactaagtgc tatgtactat tccagataaa atagtcagca aggtaaaaat 180
gtccctacct ttatgagttt taattctagc aggaaagaaa aaataatcac ataatataca 240
gatatgcagt attaggtggt gattcttgct atgaagatac aaatgaagaa agctgagtaa 300
ggggatagag aaacaagggc ttaccagtag taaatttcca tgcaaacagt ggcaggctaa 360
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actgtcaaag aacgaagcag rtggactggg cttatatcct gtctccttgt agtagagaat 540
attgagtctg atgtgcagac cagcagatcc tttgtgtatc acctgcacca ctccctcatc 600
ccctccataa gctcacatca ggagtaacta tgttcatgta ctgtaatgtg aactcatccc 660
ctggcaacac tttagtggtg tacagtaagc aactgactgg tctaggaaaa gaagccctag 720
ggcaaaggag agatgaattg agaacatctt ggcagggacc attttgtact atgtgaatct 780
gaaagcagag atatataaaa taatgaagat gacaggcaga gaaaattaga gataagaaat 840
atacagagga tttcttgtat aacacacagt gtactatcct tagatcaatc cttttacata 900
tcttctttgg cttctgtgag ttagcccttt gttatgataa caaattttat ctttttgatc 960
aatctaactc aggcacttgt gttccccatg atgacatgct c 1001
<210> 13
<211> 1001
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
aagaagaaag ttcatcagct tgctatgacc gtggtcagtt tccatcaggt ggattatacc 60
tttgaccgga atgtgttatc caggctgtta aatgaatgca gagagatgct gcaccaaatc 120
attcagcgcc acctcactgc caagtcacat ggacgggtta ataatgtgtt tgatcatttt 180
tcagattgtg aatttttggc tgccttgtat aatccttttg ggaattttaa accccactta 240
caaaaactat gtgatggtat caacaaaatg ttggatgaag agaacatatg agcacatgag 300
ttaagattgt gactgatcat gatttatttg aagatggagc actgctgatt tatgaaggaa 360
aaaagaagaa ttttctaaag attacacata tttcagaaag actttaccca attcagttgt 420
cagacataat gatttatttg aaggcttgtt ttatttgaag aaaagcatat tgccaaaaat 480
tctggttaaa agcttcctaa ygggtaacag accatgggag agatatgtgg ttgggtaatg 540
caaatgtagt tatacaaaga aaaatacaga tgtctccaga cctgaggact ttttaatagg 600
gcagttgttg tgttggtggc acattggata tttctaacat gtacaaagct atgtattttg 660
atttactttc atttcttgct atgtatatgt acttttctta aaatgccaag aactttctct 720
tgctatcatt gctccttttg aaacaattca attttcatgt ctacagctga ctgttttgtt 780
aagattgagt catcgacatt caggatttaa gtctgaggta gtcaaccctc aggaaaaaaa 840
aaatggctta tctgaaatca gtactgtgga aatgaactat attagctatt atgaataatg 900
tccagtataa gaatatgctt ctggaattga gttctccttt taagtaccaa tgatacttaa 960
atttctcaga aatgtaatgg tgtgtcattg ccttgaaatg c 1001
<210> 14
<211> 1001
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
gaggcagtga aattcctaga tttgaataat gtaatgactg agcatttgta gttaaataag 60
aaagaaaaga aaaacctcat tgtggtgata aaagctatcc aagaaaagca ggctgcttca 120
ctgggcaagg aattccccat ggctggagga gtttcccaga agccaggcca ccaccacaat 180
gaagcaaaga agtcaagcgt gggacggagg agggtcactc ctgaggttcc cgttaacttc 240
atcttctatg gccacatctc cattcttcat cgttttgaga attaatgatg caccaaatcc 300
cagaaatttc ttcccctttt ttcccagatg agtctaagtt gaaaaattaa cctttattta 360
tctttaccaa tccacagact agagtaactg tgctgatccc agataaacgt ccttctccta 420
tttcattagt ttaagtctct taaaattctc tttttgatat actatttacc tgatccaaaa 480
tccttcacag ttgcccaccc yctccaggct aacaccctgt cactttagcc tggcactctg 540
gaccctccac catcaggctc cagaacatca ccttttcagc ctcaccttga actgctcttt 600
atccacccgc ttggttctct actctgttaa atgcacggcc actggaacac tcctcctctt 660
ttccatttcc acattcttct ttacattatt ccagtgccag tcatatttgt agtatcttat 720
ttccttattc tttggagtat tttcttattt gcttatgctt ctggagagca tggactgtct 780
tcatcattac acagccagtg cccagtctgg cactcaagcc atgtattgat tggaatcaaa 840
ttgaattaca aacatttttt ttttcaatga gggaggcaga agagcatatt ggttaagagc 900
acaaattttg tagccagact tcctcggtgt gagtccaaac tttactattt accaacgagg 960
tggccttgat caagttactt aaactttttg gaattcagtt t 1001
<210> 15
<211> 1001
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
ctcggaggag ctccgtgatc aaggtgcaga tgcggcaggt gggccggcct caatcatgtc 60
tccaattgcg acggtgaatg cggtgaggag tttcgttggc ccatcacctc tgccttgggc 120
ctggctcact ttcactgctg agttagttcc acggccgcct ttgatgatgc cgcttcagca 180
tcttttttct tcggcgtttc ctgctccttt gttttcaagg tcactctgtc ctgcctgccc 240
cactctgggc cacccaaggc caagcctcta agctccgcgt tgctgaccac agttcttcac 300
actagcctca ctgcgaggct ccgtccagac tgaacatggg gaagatgaac ctttcaccat 360
ttgatgtctt gagcttctcc tctagaacaa gcaggctagc tgccctgctc agggccggct 420
catctctttc tccctggagg acagagctct gggccctgag tcagaactag ctggggttcc 480
tcactaactg ggtagactta hatgatgcat gagacctata gactcttact tagttttctc 540
agaaataaca taggccaaga acgcctgctt gcctgtctcc acaggctggg atgaggccct 600
tatgagacca agaatgggag agggcttcat aaactgtaaa ttaatctggg agcattacag 660
tcatggttat tagtttacag agattgagtt cacgttgtta tttgctctct tagcaccctg 720
ttctttttct tcatagcact ttcacatttt ctaatgatat tataagttat tattattact 780
aagcacgtag tttatgggtc taatgcctcc ctcctcacca gggtataagc taccgcagag 840
tggaagccat gcttcatttt gtttctcatc actctgccag cacctggttc agagcctgta 900
atgttgtggg ggtttaagga atactaacga taaactggtg agttatttga ggacccacga 960
ccatcctcgc aagagacttt tgttaggcca gtctgtcttg t 1001
Claims (6)
1. a kind of for detecting the primer of the relevant SNP site of lymph cancer neurological susceptibility, which is characterized in that the primer includes
The primer in the sites rs872071, the primer in the sites rs2647012, the primer in the sites rs1045241, the sites rs6773854 are drawn
Object and the primer in the sites rs6421571；
Wherein, the primer in the sites rs872071 is F：5 '-GAACAACTCTGGGAGTCT-3 ' and R：5′-
CTCTCTGAAGGAACGTAAC-3′；
The primer in the sites rs2647012 is F：5 '-GATATGCAGTATTAGGTGGT-3 ' and R：5′-
TAGACCAGTCAGTTGCTTAC-3′；
The primer in the sites rs1045241 is F：5 '-GTGGATTATACCTTTGACC-3 ' and R：5′-
GTCGATGACTCAATCTTAAC-3′；
The primer in the sites rs6773854 is F：5 '-CTTTACCAATCCACAGACTA-3 ' and R：5′-
GCTGTGTAATGATGAAGAC-3′；
The primer in the sites rs6421571 is F：5 '-CTTTCACTGCTGAGTTAGTT-3 ' and R：5′-
CTCACCAGTTTATCGTTAGT-3′。
2. using the method for the relevant SNP site polymorphism of primer detection lymph cancer neurological susceptibility described in claim 1, feature
It is, includes the following steps：
(1) it acquires sample and extracts its genomic DNA；
(2) it carries out the PCR amplification of target gene to genomic DNA respectively using the primer, and glue is carried out to amplified production and is returned
It receives；
(3) concentration mensuration is carried out to glue recovery product, then calculation template amount carries out fluorescent marker PCR reactions, and produced to reaction
Object is purified；
(4) machine in the purified product in step (3) is sequenced, and SNP testing results is analyzed.
3. the method for the detection relevant SNP site polymorphism of lymph cancer neurological susceptibility according to claim 2, feature exist
In PCR amplification system is in step (2)：DNA profiling content is 100-150ng, 3.0 μ of forward primer of a concentration of 5pmol/ μ L
3.0 μ L, Prime STAR MAX of reverse primer, the 25.0 μ L of L, a concentration of 5pmol/ μ L, use ddH2O supplies volume to 50 μ L.
4. the method for the detection relevant SNP site polymorphism of lymph cancer neurological susceptibility according to claim 2, feature exist
In pcr amplification reaction condition is in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 15s, 72 DEG C extend
15s carries out 30 cycles, last 72 DEG C of extensions 5min.
5. the method for the detection relevant SNP site polymorphism of lymph cancer neurological susceptibility according to claim 2, feature exist
In PCR amplification system is in step (3)：DTCS Master Mix 2.5 μ L, 1.0 μ of reverse primer of a concentration of 5pmol/ μ L
L, DNA profiling 20ng, uses ddH2O supplies volume to 10 μ L.
6. the method for the detection relevant SNP site polymorphism of lymph cancer neurological susceptibility according to claim 2, feature exist
In pcr amplification reaction condition is in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C of annealing 25s, 60 DEG C extend
3min, 30 cycles, last 60 DEG C of extensions 20min.
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