CN108531601A - A kind of primer and detection method for detecting the relevant SNP site of liver cancer susceptibility - Google Patents
A kind of primer and detection method for detecting the relevant SNP site of liver cancer susceptibility Download PDFInfo
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Abstract
The present invention provides a kind of primers for detecting the relevant SNP site of liver cancer susceptibility, which includes the primer in the sites rs230496, the primer in the sites rs200842482, the primer in the sites rs4939827, the primer in the sites rs7240004 and the primer in the sites rs7229639.Using the method for the relevant SNP site polymorphism of the primer detection liver cancer susceptibility, include the following steps：(1) it acquires sample and extracts DNA；(2) PCR amplification of target gene is carried out using primer respectively, glue recycles；(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 liver cancer.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of for detecting the relevant SNP site of liver cancer susceptibility
Primer and detection method.
Background technology
Primary carcinoma of liver (referred to as " liver cancer ") be by liver cell or the cytogenetic malignant tumour of intrahepatic biliary epithelium, due to
Most liver cancer derive from liver cell, so usually said liver cancer refers to hepatocellular carcinoma more.China's tumour Entry year in 2017
Report display arranges the 3rd in male malignancy morbidity in urban area liver cancer, the 6th is arranged in female malignant morbidity,
And the death rate of liver cancer occupies the 3rd in small and medium-sized cities, and the 2nd is occupied in big city.Since onset of liver cancer is hidden, initial stage does not face
Bed symptom, most of patients have missed best occasion for the treatment when medical.Therefore, the early prediction, early prevention of liver cancer just seem extremely heavy
It wants.At present it is believed that Risk Factors of Liver Cancer include inherent cause, hepatites virus infections, aflatoxin exposure, drink and
Smoking, contaminated drinking water and liver fluke infection etc..Further investigation is found, the difference in above-mentioned risk factor
Body has the difference for occurring and liver cancer not occurring.It is what results in this species diversityWhy onset of liver cancer divides between both sexes
Cloth unevenness (male's more susceptible disease)Why the morbidity of liver cancer has certain Familial aggregation phenomenonThese phenomenons prompt us, liver
The morbidity of cancer largely additionally depends on the genetic predisposition of individual.Gene polynorphisms are to cause disease that inheritance susceptible occurs
The importance of property, wherein most commonly seen with single nucleotide polymorphism (Single Nucleotide Polymorphsm, SNP).
SNP refers to the DNA sequence polymorphism caused by a single nucleotide variation in genomic level, it is that the mankind are heritable
The most common type in genome mutation accounts for 90% or more of all known polymorphisms.SNP is deposited extensively in human genome
Just there is 1 in average every 500-1000 base-pair, is estimating that its sum is even more up to 3,000,000.
So far, this field is not found to be the detection of liver cancer susceptible risk and provides one group of closely related tumor susceptibility gene and draw
Object.
Invention content
For the above-mentioned problems in the prior art, the present invention provides a kind of relevant for detecting liver cancer susceptibility
The primer and detection method of SNP site, primer specificity is good, and detection method accuracy is high, can be used for the risk assessment of liver cancer,
Guidance is provided for the disease prevention of liver 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 liver cancer susceptibility, these primers include NFKB1 genes
The sites rs4939827 of the primer in the sites rs230496, the primer in the sites rs200842482 of XRCC1 genes, SMAD7 genes
Primer, SMAD7 genes the sites rs7240004 primer and SMAD7 genes the sites rs7229639 primer；
Wherein, the primer in the sites rs230496 is F：5′-CCAGCAAAGGTTATTGTTCAGT-3′(SEQ ID No：1)
And R：5′-AAAGCCCTACATATTCACACC-3′(SEQ ID No：2)；
The primer in the sites rs200842482 is F：5′-GCTCCTCTCAGTAGTCT-3′(SEQ ID No：And R 3)：5′-
CCAAGTATCGGCCAGA-3′(SEQ ID No：4)；
The primer in the sites rs4939827 is F：5′-GACAAGCCTAAGATAAAAGG-3′(SEQ ID No：And R 5)：5′-
GATTCATCGTCTGATCTGT-3′(SEQ ID No：6)；
The primer in the sites rs7240004 is F：5′-GCTTAGAATGTTTCAGCAC-3′(SEQ ID No：And R 7)：5′-
CTTGTGTCTGATGTGTAGG-3′(SEQ ID No：8)；
The primer in the sites rs7229639 is F：5′-CTGAGATCTGTAGCAGGAC-3′(SEQ ID No：And R 9)：5′-
TTGTGTGTGCTTAGAGATAG-3′(SEQ ID No：10).
Using the method for the relevant SNP site polymorphism of above-mentioned primer detection liver cancer 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 partings are 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, mended with ddH2O
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 detecting the primer and detection method of the relevant SNP site of liver cancer susceptibility, have following
Advantageous effect：
(1) the present invention provides the primer for detecting liver cancer susceptibility related locus, the primer have specificity it is high,
The good advantage of accuracy, realizes the detection of the relevant SNP site of liver cancer susceptibility, improves detection efficiency.
(2) the present invention also provides a kind of method of the detection relevant SNP site of liver cancer susceptibility, this method is directly to survey
The advantages that sequence method, this detection method is simple, and testing result also has specificity good, high sensitivity, accuracy is good, can be liver
The disease prevention of 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 rs230496 loci polymorphisms detection；
Fig. 3 is the sequencing result figure of rs200842482 loci polymorphisms detection；
Fig. 4 is the sequencing result figure of rs4939827 loci polymorphisms detection；
Fig. 5 is the sequencing result figure of rs7240004 loci polymorphisms detection；
Fig. 6 is the sequencing result figure of rs7229639 loci polymorphisms detection；
Fig. 7 is the fluorescence proof diagram of rs230496 loci polymorphism testing results；
Fig. 8 is the fluorescence proof diagram of rs200842482 loci polymorphism testing results；
Fig. 9 is the fluorescence proof diagram of rs4939827 loci polymorphism testing results；
Figure 10 is the fluorescence proof diagram of rs7240004 loci polymorphism testing results；
Figure 11 is the fluorescence proof diagram of rs7229639 loci polymorphism testing results.
Specific implementation mode
The primer of the design amplification SNP site of embodiment 1
Devise a large amount of primer for the relevant SNP site of liver cancer susceptibility, by the optimization of primer reaction condition and
Compare, filters out five pairs of good primers of specificity, including：
The primer in the sites rs230496 of NFKB1 genes is F：5′-CCAGCAAAGGTTATTGTTCAGT-3′(SEQ ID
No：And R 1)：5′-AAAGCCCTACATATTCACACC-3′(SEQ ID No：2)；
The primer in the sites rs200842482 of XRCC1 genes is F：5′-GCTCCTCTCAGTAGTCT-3′(SEQ ID
No：And R 3)：5′-CCAAGTATCGGCCAGA-3′(SEQ ID No：4)；
The primer in the sites rs4939827 of SMAD7 genes is F：5′-GACAAGCCTAAGATAAAAGG-3′(SEQ ID
No：And R 5)：5′-GATTCATCGTCTGATCTGT-3′(SEQ ID No：6)；
The primer in the sites rs7240004 of SMAD7 genes is F：5′-GCTTAGAATGTTTCAGCAC-3′(SEQ ID
No：And R 7)：5′-CTTGTGTCTGATGTGTAGG-3′(SEQ ID No：8)；
The primer in the sites rs7229639 of SMAD7 genes is F：5′-CTGAGATCTGTAGCAGGAC-3′(SEQ ID
No：And R 9)：5′-TTGTGTGTGCTTAGAGATAG-3′(SEQ ID No：10).
PCR amplification is carried out respectively using the primer pair testing gene group DNA of the relevant SNP site of liver cancer susceptibility, and PCR expands
Increasing system is：DNA profiling x μ L make its content for 100-150ng, deionized water 19-x μ L, 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 1,2,3 holes are rs230496
The primer amplification band in site；4,5,6 holes are the primer amplification band in the sites rs200842482；7,8,9 holes are rs4939827
The primer amplification band in site；10,11,12 holes are the primer amplification band in the sites rs7240004；13,14,15 holes are
The primer amplification band in the sites rs7229639.
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 liver cancer 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 above-mentioned rs230496, rs200842482, rs4939827, the sites rs7240004 and rs7229639
Primer respectively to the DNA of extraction carry out PCR amplification.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 | a(100-150ng) |
| ddH2O | 19-a |
| 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, 96 hole sample panels, 96 hole buffer solutions and glue groove are finally packed into the full-automatic sequenators of ABI3730
It is sequenced.
By sequence analysis software (ABI3730 Chromas), sequencing result is compared with standard sequence, is found
SNP site, by the type for analyzing 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 rs230496 loci polymorphisms detection in Fig. 2, and genotype is followed successively by
AA, GG and AG；Wherein AA is the non-susceptible genotype in the sites rs230496, and the tumor susceptibility gene that GG and AG is the sites rs230496
Type.
3-1,3-2 and 3-3 are the sequencing result figure of rs200842482 loci polymorphisms detection in Fig. 3, and genotype is successively
For CC, TT and CT；Wherein CC is the non-susceptible genotype in the sites rs200842482, and TT and CT is the sites rs200842482 position
The susceptible genotype of point.
4-1,4-2 and 4-3 are the sequencing result figure of rs4939827 loci polymorphisms detection in Fig. 4, and genotype is followed successively by
CC, TT and CT；Wherein CC is the non-susceptible genotype in the sites rs4939827, and the easy sensillary base that TT and CT is the sites rs2274223
Because of type.
5-1,5-2 and 5-3 are the sequencing result figure of rs7240004 loci polymorphisms detection in Fig. 5, and genotype is followed successively by
GG, AA and AG；Wherein GG is the non-susceptible genotype in the sites rs7240004, and the easy sensillary base that AA and AG is the sites rs7240004
Because of type.
6-1,6-2,6-3 are the testing result figure of the sites rs230496 fluorescence probe method in Fig. 6, and genotype is followed successively by
AA, GG and AG；Wherein AA is the non-susceptible genotype in the sites rs230496, and the tumor susceptibility gene that GG and AG is the sites rs230496
Type.
Embodiment 3 detects the specificity of the relevant SNP site of liver cancer susceptibility
This detection method by specificity be defined as sequencing result peak figure without set peak, background peaks, miscellaneous peak, float peak phenomena such as.
It is detected according to 30 samples of detection method pair provided by the invention, sequencing peak figure is single, without bimodal, background peaks
Phenomena such as, 30 sample detection result all sames illustrate that detection method specificity provided by the invention is 100%.
Embodiment 4 detects the sensitivity and accuracy of the relevant SNP site of liver cancer susceptibility
Sensitivity definition is testing result coincidence rate by this detection method.
It is detected according to 50 samples of detection method pair provided by the invention, while being tested using fluorescence probe method
Card, the testing result of 50 samples of two methods pair is consistent, and the accuracy of this detection method has reached 100%, further verifies
High using the sensitivity and accuracy of PCR sequencing PCR detection SNP site polymorphism, result is as shown in Fig. 7-Figure 11.
Wherein, in Fig. 7 7-1,7-2,7-3 be rs230496 loci polymorphism testing results fluorescence proof diagram, gene
Type is followed successively by AA, GG and AG；Wherein AA is the non-susceptible genotype in the sites rs230496, and GG and AG is the sites rs230496
Susceptible genotype.
8-1,8-2 and 8-3 are the fluorescence proof diagram of rs200842482 loci polymorphism testing results, genotype in Fig. 8
It is followed successively by GG, AA and AG；Wherein GG is the non-susceptible genotype in the sites rs200842482, and AA and AG is rs200842482
The susceptible genotype in point site.
9-1,9-2 and 9-3 are the fluorescence proof diagram of rs4939827 loci polymorphism testing results in Fig. 9, genotype according to
Secondary is CC, TT and CT；Wherein CC is the non-susceptible genotype in the sites rs4939827, and TT and CT is the easy of the sites rs2274223
Feel genotype.
Figure 10-1,10-2 and 10-3 are the fluorescence proof diagram of rs7240004 loci polymorphism testing results, genotype base
Because type is followed successively by GG, AA and AG；Wherein GG is the non-susceptible genotype in the sites rs7240004, and AA and AG is rs7240004
The susceptible genotype of point.
Figure 11-1,11-2 and 11-3 are the fluorescence proof diagram of rs7229639 loci polymorphism testing results, genotype according to
Secondary is GG, AA and AG；Wherein GG is the non-susceptible genotype in the sites rs7229639, and AA and AG is the easy of the sites rs7229639
Feel genotype.
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 liver cancer susceptibility
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Claims (6)
1. a kind of primer for detecting the relevant SNP site of liver cancer susceptibility, which is characterized in that the primer includes NFKB1
The primer in the sites rs230496 of gene, the primer in the sites rs200842482 of XRCC1 genes, SMAD7 genes
The positions rs7229639 of the primer in the sites rs4939827, the primer in the sites rs7240004 of SMAD7 genes and SMAD7 genes
The primer of point；
Wherein, the primer in the sites rs230496 is F：5 '-CCAGCAAAGGTTATTGTTCAGT-3 ' and R：5′-
AAAGCCCTACATATTCACACC-3′；
The primer in the sites rs200842482 is F：5 '-GCTCCTCTCAGTAGTCT-3 ' and R：5′-CCAAGTATCGGCCAGA-
3′；
The primer in the sites rs4939827 is F：5 '-GACAAGCCTAAGATAAAAGG-3 ' and R：5′-
GATTCATCGTCTGATCTGT-3′；
The primer in the sites rs7240004 is F：5 '-GCTTAGAATGTTTCAGCAC-3 ' and R：5′-
CTTGTGTCTGATGTGTAGG-3′；
The primer in the sites rs7229639 is F：5 '-CTGAGATCTGTAGCAGGAC-3 ' and R：5′-
TTGTGTGTGCTTAGAGATAG-3′。
2. using the method for the relevant SNP site polymorphism of primer detection liver cancer susceptibility described in claim 1, feature exists
In including 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 liver cancer susceptibility according to claim 2, which is characterized in that
PCR amplification system is in step (2)：DNA profiling content be 100-150ng, the 3.0 μ L 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 a concentration of 5pmol/ μ L, volume is supplied to 50 μ L with ddH2O.
4. the method for the detection relevant SNP site polymorphism of liver cancer susceptibility according to claim 2, which is characterized in that
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 liver cancer susceptibility according to claim 2, which is characterized in that
PCR amplification system is in step (3)：The 1.0 μ L of reverse primer of DTCS Master Mix 2.5 μ L, a concentration of 5pmol/ μ L,
DNA profiling 20ng, uses ddH2O supplies volume to 10 μ L.
6. the method for the detection relevant SNP site polymorphism of liver cancer susceptibility according to claim 2, which is characterized in that
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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| US20160102358A1 (en) * | 2014-10-14 | 2016-04-14 | Wake Forest University Health Sciences | Methods and compositions for correlating genetic markers with cancer risk |
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