CN104232768B - Colorectal cancer susceptibility diagnostic kit and the application of SNP in its preparation - Google Patents
Colorectal cancer susceptibility diagnostic kit and the application of SNP in its preparation Download PDFInfo
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Abstract
The invention discloses a kind of diagnostic kit and the purposes of SNP in the preparation of this test kit of colorectal cancer susceptibility, involved SNP site is rs12522693 and rs17836917.Contriver is by genome-wide association study, and experience screening stage, first stage checking and subordinate phase demonstrate rs12522693 site G allelotrope and the significant correlation between rs17836917 site A allelotrope and colorectal cancer susceptibility.Accordingly by the genotype detecting rs12522693 and/or rs17836917 site, the colorectal cancer susceptibility of experimenter can be predicted, thus early prevention and the treatment of disease can be promoted.The diagnostic kit of colorectal cancer susceptibility can utilize technology known in the art to detect the genotype in rs12522693 and/or rs17836917 site, judges whether experimenter has colorectal cancer susceptibility.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of colorectal cancer susceptibility diagnostic kit and the purposes of SNP in preparation colorectal cancer susceptibility diagnostic kit.
Background technology
Colorectal cancer is the common cancer of masculinity and femininity the 3rd, points out 2012 annual U.S. colorectal carcinoma new cases 103170 in american cancer statistical reports in 2013, and the rectum cancer reaches 40290 examples, the two mortality ratio 50830.And in China, along with the change of mode of life and food habits in recent years, Colorectal Cancer and death rise all fast, according to statistical result showed in 2011, colorectal cancer occupies China's Cancer Mortality the 3rd, be the 4th and cause dead malignant tumour, M & M, all close to contemporaneously world standard, becomes the public health problem of serious threat crowd life and health.The pathogenesis of colorectal cancer is illustrated not yet completely, but Study of Etiology shows, most colorectal cancer be a multi-step, multistage, environmental factors and the coefficient complex process of inherited genetic factors.Although research has shown the importance of environmental factors for Colorectal Cancer, but contact identical environmental risk factors but only have only a few individuality occur tumour, illustrate that environmental factors is the initiating agent that colorectal cancer occurs, the hereditary feature of individuality then determines the susceptibility to colorectal cancer.Therefore, systematic research is carried out to colorectal cancer inheritance susceptible factor, find colorectal cancer excessive risk gene or site, for realizing the early diagnosis of colorectal cancer and effectively preventing that there is great practical significance.
Gene pleiomorphism refers to the difference of genome sequence between the Different Individual in different plant species or same species, these differences cause because DNA allelotrope nucleotide in karyomit(e) changes, and mainly comprises the change of the replacement of base, insertion, disappearance and tumor-necrosis factor glycoproteins copy number.Single nucleotide polymorphism (SingleNucleotidePolymorphism, SNP) be the class genetic marker system proposed by the scholar Lander (1996) in the human genome research centre of Massachusetts Institute Technology, just refer to the polymorphism caused due to the replacement of single core thuja acid (A/T/C/G) in genomic dna sequence.Its variant form has: transversion, conversion, insertion and disappearance etc., caused by the conversion of single base or transversion.The SNPs with the nucleotide variation of conversion hysteria accounts for 2/3.SNPs may regulate and control the expression of encoding gene or Noncoding gene at transcriptional level (as affected the combination of transcription factor) or post-transcriptional level (regulatory mechanism as miRNA), and then affect related biological function, thus cause the difference of numerous proterties between individuality, comprising the difference of disease susceptibility.
The individuality of different genetic background is under identical environmental exposure, and the susceptibility of colorectal cancer is also different, and this susceptibility is considered to closely related with the inherited genetic factors of individuality, and wherein major part is also caused by SNPs.Therefore associate by what explore SNPs and colorectal cancer susceptibility in genome the susceptible mark finding colorectal cancer further, there is potential biology and clinical meaning.
As the Human Genome Project (HumanGenomeProject, HGP) and international human genome haplotype figure plan (IntemationalHapMapProject) and high-throughput genotyping technique in conjunction with time, genome-wide association study (Genome-WideAssociationStudy, GWAS) develops the effective tool of the inheritance susceptible mark into finding complex disease or proterties rapidly.Genome-wide association study is the research and design adopting case-control, utilize HapMap and thousand human genome plan (1000genomeproject) databases, with millions of single nucleotide polymorphism SNP for mark carries out case and contrast association analysis, and be aided with multiple independently research carry out subsequent authentication, finishing screen selects the heritable variation with this disease significant correlation.Compared with traditional candidate gene strategy, the SNPs site of GWAS covers whole genome area, without the need to building Research Hypothesis in advance, through primary dcreening operation and multistage individual authentication, statistics usefulness is higher, and experimental result is reliable, more truly can disclose the inner link between disease and inherited genetic factors.
Summary of the invention
Contriver has found the dependency of rs12522693 site G allelotrope and rs17836917 site A allelotrope and colorectal cancer, and develop the diagnostic kit of colorectal cancer susceptibility accordingly, and rs12522693 site G allelotrope and the application of rs17836917 site A allelotrope in the test kit preparing Colorectal Cancer Diagnosis susceptibility.
On the one hand, the invention provides two SNP site for Colorectal Cancer Diagnosis susceptibility, described SNP site is rs12522693 and rs17836917, when rs12522693 loci gene type is GG, judge individual as colorectal cancer susceptibility individuality, when rs17836917 loci gene type is AA, judge individual as colorectal cancer susceptibility individuality.
On the other hand, the invention provides for detecting the genotypic reagent of SNP site, described reagent can be the reagent adopting any technology for detection SNP known in the art, as long as it can detect rs12522693 and/or rs17836917 loci gene type in sample.
In a specific embodiment of the present invention, detect the genotypic reagent of SNP site and comprise the reagent used needed for SequenomMassArray detection SNP site genotype, the described use SequenomMassArray reagent detected needed for SNP site genotype comprises the primer of the nucleotide sequence for the SNP site that increases.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.Preferably, detect the extension primer that the genotypic reagent of SNP site also comprises the reaction of single base amplification, for rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
In another specific embodiment of the present invention, detect the genotypic reagent of SNP site and comprise the reagent used needed for direct sequencing detection SNP site genotype.Mentioned reagent comprises the primer of the nucleotide sequence for the SNP site that increases, for rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.
Another aspect, the invention provides for detecting the genotypic test kit of SNP site, this test kit can comprise the reagent adopting any technology for detection SNP known in the art, as long as it can detect rs12522693 and/or rs17836917 loci gene type in sample.
In a specific embodiment of the present invention, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the primer of the nucleotide sequence for the SNP site that increases.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.
In an alternative embodiment, test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer of single base amplification reaction, for rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
In the embodiment that another is alternative, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer for the primer of the nucleotide sequence of the SNP site that increases and the reaction of single base amplification.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.For rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
Another aspect, the invention provides the test kit for Colorectal Cancer Diagnosis susceptibility, this test kit can comprise the reagent adopting any technology for detection SNP known in the art, as long as it can detect rs12522693 and/or rs17836917 loci gene type in sample.
In a specific embodiment of the present invention, the test kit for Colorectal Cancer Diagnosis susceptibility comprises the primer of the nucleotide sequence for the SNP site that increases.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.
In an alternative embodiment, the test kit for Colorectal Cancer Diagnosis susceptibility comprises the extension primer of single base amplification reaction, and for rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
In the embodiment that another is alternative, the test kit for Colorectal Cancer Diagnosis susceptibility comprises the extension primer for the primer of the nucleotide sequence of the SNP site that increases and the reaction of single base amplification.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.For rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
Preferably, test kit for detecting rs12522693 and/or rs17836917 loci gene type of the present invention and also comprise DNA extraction reagent for the test kit of Colorectal Cancer Diagnosis susceptibility, DNA extraction reagent comprises phenol, chloroform, primary isoamyl alcohol, ethanol.
Another aspect, the invention provides a kind of method detecting rs12522693 and/or rs17836917 loci gene type.The operation steps of described detection method is as follows:
(1) genomic dna is extracted;
(2) SequenomMassArray detection method or direct sequencing is used to detect rs12522693 and/or rs17836917 loci gene type.Use in SequenomMassArray detection method for the SNP site that increases at the primer of interior nucleotide sequence and the extension primer that reacts for single base amplification; Use in direct sequencing for the primer of SNP site at interior nucleotide sequence that increase.
What in step (2), SequenomMassArray detection method used is as follows at the primer sequence of interior nucleotide sequence for the SNP site that increases: for rs12522693 site, forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.The extension primer sequence reacted for single base amplification that SequenomMassArray detection method uses is as follows: for rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
What in step (2), direct sequencing used is as follows at the primer sequence of interior nucleotide sequence for the SNP site that increases: for rs12522693 site, forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.Sequencing primer is the reverse primer in amplimer.
Present invention also offers the purposes of reagent in the genotypic test kit of preparation detection SNP site detecting rs12522693 and/or rs17836917 loci gene type, described test kit can comprise the reagent adopting any technology for detection SNP known in the art, as long as it can detect rs12522693 and/or rs17836917 loci gene type in sample.In a specific embodiment of the present invention, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the primer of the nucleotide sequence for the SNP site that increases.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.In an alternative embodiment, test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer of single base amplification reaction, for rs12522693 site, described extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.In the embodiment that another is alternative, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer for the primer of the nucleotide sequence of the SNP site that increases and the reaction of single base amplification.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.For rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
The reagent that present invention also offers for detecting rs12522693 and/or rs17836917 loci gene type is applied in the test kit for the preparation of Colorectal Cancer Diagnosis susceptibility, described test kit can be the reagent of employing any technology for detection SNP known in the art, as long as it can detect rs12522693 and/or rs17836917 loci gene type in sample.In a specific embodiment of the present invention, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the primer of the nucleotide sequence for the SNP site that increases.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.In an alternative embodiment, test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer of single base amplification reaction, for rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.In the embodiment that another is alternative, the test kit for detecting rs12522693 and/or rs17836917 loci gene type comprises the extension primer for the primer of the nucleotide sequence of the SNP site that increases and the reaction of single base amplification.For rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.For rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
Accompanying drawing explanation
Fig. 1 represents the schematic diagram of GWAS blood sample white corpuscle extraction step;
Fig. 2 represents DNA quality control standard figure;
Fig. 3 represents TapMan gene type schematic diagram, wherein black side's point represents NTC, red (leftmost point) represents wild homozygote, and blue (middle point) represents heterozygote, and green (rightmost point) represents no mutant homozygote;
Fig. 4 represents that the GWAS primary dcreening operation stage is at P < 10
-4time there is the Manhattan figure in the SNPs site of significant difference, wherein P value is the P value of SNPs under additive model under logistic regression model after age, sex and first principal component adjust; Ordinate zou represent P value take the logarithm after negative value (-log
10p), X-coordinate represents the relative position of SNPs in karyomit(e), and horizontal line represents got threshold value (1 × 10
-4).
Concrete embodiment
Full-length genome screening stage adopts AffymetrixAxiom
tMchip platform, first stage checking adopts SequenomMassArray platform, and subordinate phase checking adopts TaqMan gene type platform.
First in each array, the genomic dna of 250ng is decomposed by restriction endonuclease Nsp1 or Sty1, then by identifying that the concatenator of cohesive force between four bases reconnects, carries out pcr amplification.DNA after amplification is detected by agarose gel electrophoresis, can verify and obtain the fragmentation DNA that mean length is 250bp to 1500bp, and carry out drying, resuspended, purifying.The DNA deoxyribonuclease I getting 90 μ g purifying digests further, then carries out with the sepharose of 4% checking that fragment is less than the product of 180bp.The DNA product corresponding to Nsp and Sty by vitamin H carries out end mark, and sample is merged hybridization.Carry out stringency washes after crossover process completes, non-specific background can be removed and specific binding is retained.The polychrome coupled reaction that each SNP is occurred by chip surface is differentiated.After coupled reaction completes, chip will complete the steps such as dyeing, washing on GeneTitan hyperchannel automatization chip operation station, adopt AffymetrixGTC4.1 software by scan sample data importing, the fluorescent signal of each sample is changed into typing data.
SequenomMassARRAYSNP testing process is in conjunction with multiple PCR technique, MassARRAYiPLEX single-basic extension technology, somatotype detection is carried out with matrix solid-dispersion flying time mass spectrum analysis mass-spectrometric technique (matrix-assistedlaserdesorption/ionization-timeofflight, MALDI-TOF).The DNA profiling comprising SNP site region is increased by round pcr, re-uses special extension primer and PCR primer carries out single base extension.Because pleomorphism site base is different, the different terminal bases of extension products will cause the difference of the molecular weight of product after extending, therefore the base difference caused by SNP polymorphism is embodied by the difference of molecular weight, by matrix solid-dispersion flying time mass spectrum analysis mass-spectrometric technique, detect the size of extension products molecular weight, the analysis software of application specific, carries out the detection of SNP somatotype by judging the difference of molecular weight.
TaqMan allelic gene typing technology is the SNP typing method of American AB I company research and development, is one of standard of the gene type of comparatively generally acknowledging at present.Cardinal principle: add a pair two ends has different fluorescently-labeled specific probe to identify different allelotrope when PCR reacts, 5 ' end of probe sequence is reporter fluorescence group (reporter), and 3 ' end is quenching fluorescence group (quencher).In PCR process, two probes can be combined by the Annealing complementary special with template allelic sequences.Now probe sequence is complete, and reporter fluorescence group is quenched fluorophor suppression and no signal sends.When subchain copies to probe and template junction, archaeal dna polymerase plays 5 '-3 ' 5 prime excision enzyme activity, cut away reporter fluorescence group make its depart from 3 ' end quenching go out fluorophor cancellation effect thus send fluorescence.5 ' end of two probes indicates different fluorescence (FAM, VIC or HEX), and 3 ' end indicates MGB quenching group combination.According to the different fluorescence detected, the SNP allelotype of corresponding sample can be judged.
The qualification in embodiment 1 Human Colorectal Cancer susceptibility genes involved group SNPs site
1. research object
This research is divided into GWAS primary dcreening operation stage, first stage checking and subordinate phase to verify three parts.The sample (mainly from The People's Hospital of Peking University and tumour hospital of Peking University) of GWAS primary dcreening operation stage employing Beijing area, case load is 932 examples, and control group is 1006 examples.First stage checking adopts the sample of Central China (Jiangsu and Shanghai), comprises 1759 routine cases and 1875 example contrasts.Subordinate phase checking contains 943 routine cases from Beijing, Shandong and the Northeast and 1838 example contrasts.All cases are the Chinese Han Population of originating based on hospital, through the colorectal cancer that histopathology or cytology are clarified a diagnosis.Control group from the healthy population sample of local health examination and Chinese Marrow Donor Program data bank, and mates with case according to age-sex and area.
2. experimental procedure
The collection of 2.1 blood specimens and the extraction of genomic dna
Full-fledged research object all gathers with vacuum EDTA anticoagulant blood-collecting pipe and plays peripheric venous blood 5ml on an empty stomach morning, the centrifugal 10min of 3000rpm/min, separated plasma, leukocytic cream and red corpuscle.Lymphocyte separation medium can be used to be extracted by granulocyte further as having ready conditions; As unconditionally, then directly leukocytic cream can be sub-packed in 2ml cryopreservation tube, it is for subsequent use to put-80 DEG C of refrigerated storages, and detailed step as shown in Figure 1.
Adopt phenol-chloroform method to extract genomic dna, adopt ultraviolet spectrophotometry (260/280 ratio) to determine DNA purity, OD
260measure DNA concentration, after unified markization ,-20 DEG C of storages are for subsequent use.Concrete steps are as follows:
(1) leukocyte suspension is moved into 5ml centrifuge tube, add hemolyzing reagent, the centrifugal 10min of 4000rpm/min after vibration mixing, abandons supernatant, repeats this process once;
(2) add 1ml extract in precipitation, add 8 μ l Proteinase Ks after mixing, 37 DEG C of water-baths are spent the night;
(3) slightly cool after taking out, add the saturated phenol of 1mlTris-, turn upside down the centrifugal 10min of mixing 15min, 4000rpm/min;
(4) carefully draw supernatant, add the sodium-acetate 60 μ l of 3MpH5.0, then add isopyknic primary isoamyl alcohol with supernatant liquor, visible white flocculent precipitate after jog, the centrifugal 2min of 10000rpm/min;
(5) add 75% ethanol in precipitation and be about 1ml, the centrifugal 2min of 8000rpm/min, abandons supernatant;
(6) add dehydrated alcohol in precipitation and be about 1ml, the centrifugal 2min of 8000rpm/min, dry after abandoning supernatant;
(7) desciccate adds TE damping fluid 100 μ l and dissolves, by measuring OD
260/280and OD
260/230ratio determines DNA purity, and gDNA mean size is estimated by 1% agarose gel electrophoresis.-20 DEG C of storages after markization;
(8) DNA quality control standard: band is clear, length > 10kb, without obvious degradation (shown in Fig. 2); OD
260/280between 1.8 ~ 2.0, OD
260/230namely > 1.5 meets chip detection standard.
2.2 genetic polymorphism detection
2.2.1 the gene type of full-length genome chip
The gene type of the full-length genome screening stage of this research adopts AffymetrixAxiom
tMhumanCHB1 & 2 chip detects 973 routine colorectal cancer cases and 1006 example contrasts.This chip-derivation is in HapMap, dbSNP and 1000GenomesProject database, comprise the mark (referring to http://www.affymtrix.com) more than 1296521 heritable variations, the detection of full-length genome chip completes in Beijing Boao Biological Co., Ltd.With P < 1.0 × 10
-4for threshold value, next step checking is carried out in the SNPs site that the examination stage meets the demands.
2.2.2SequenomMassArray gene type
According to the statistical result in primary dcreening operation stage, select the significant SNPs site of significant difference and adopt SequenomMassArray platform to carry out first stage checking.
Concrete operation step is as follows:
(1) amplification comprises the region of DNA territory of target site;
(2) use shrimp alkaline phosphotase SAP remove system middle reaches from dNTP, purified pcr product;
(3) single base extension: the PCR primer after SAP process carries out the extension of base specific, there is difference in not homoallelic extension products molecular weight, namely the difference of extension products molecular weight embodies the different bases of pleomorphism site;
(4) resin purification: extension product adopts resin to carry out purifying and desalts;
(5) purified product is transferred on 384 hole SpectroChip, carries out MALDI-TOF mass spectroscopy, and TyperV4.0 software output detections result, determines gene type.
Subordinate phase checking can be carried out when the SNPs site result of first stage checking meets P < 0.05.
2.2.3TaqMan gene type
2.2.3.1 probe and primer
Use primer and the probe of PrimerExpress primer-design software (http://appliedbiosystems.com) custom design candidate SNP locus.
2.2.3.2 real-time quantitative PCR amplification
ABI7900HT type quantitative real time PCR Instrument is used to carry out gene type.5 μ l reaction systems in the reaction plate of every block 384 hole, containing MasterMix2.0 μ l, each 0.25 μ l of forward and reverse primer, each 0.125 μ l of TaqMan probe, DNA sample 0.5 μ l (concentration of specimens > 5ng/ μ l), deionized water 1.25 μ l.Adopt high-transmittance epiphragma to cover Sptting plate, of short duration centrifugal de-soak, load on ABI7900HT quantitative real time PCR Instrument and carry out somatotype detection.Reaction conditions is:
After amplification cycles terminates, read the rear fluorescent signal of amplification, use SDS2.0 software to carry out genotype interpretation, genotyping result as shown in Figure 3.
3. result
3.1 genome-wide association study primary dcreening operation results
(1) SNPs site is filtered and is rejected
SNPs site is filtered and is usually taked following standard, as shown in Table 1 below:
Table 1SNPs site quality control flow
(2) research object is filtered and is rejected
The filtration of research object is rejected and generally will be considered following problem: guarantee that each genes of individuals somatotype success ratio is greater than 95%; The sex result differentiated by somatotype information in site on X chromosome and the sex that provides of baseline visit are contrasted, and reject agnogenio and sex is inconsistent individuality; By rejecting the sample that heterozygosis rate departs from more than population mean heterozygosis rate 6 standard deviations to the heterozygosis rate analysis of each individuality; Reject the potential individuality with obvious population stratification, result is as shown in table 2.
Table 2 research object quality control process
Through Data Control, the SNPs on 1902 research objects (932 routine colorectal cancer cases of Beijing area and 970 example contrasts) and 1140565 euchromosomes enters GWAS data analysis.Comparatively the low bulk factor (λ=1.026) illustrates only in faint population stratification in two groups of research objects, less to the Influence on test result of association analysis.P value in scatter diagram is the P value (shown in Fig. 4) of SNPs under the additive model under logistic regression model after age, sex and first principal component adjustment.The SNPs choice criteria in examination stage is (1) SNPsP < 1.0 × 10
-4; (2) somatotype is clear; (3) if having multiple SNPs to there is strong linkage disequilibrium (LD) get P value the lowest.Result has 63 SNPs conformance with standard and enters first stage checking.
3.2 first stage the results
63 sites that first stage is selected another independently sample verify in (comprising 1759 routine cases and 1876 example contrasts), adopt the logistic regression analysis under addition genetic model (additivemodel), select the SNPs wherein with statistical significance (P < 0.05).Result has 7 SNPs to have statistical significance, enters the checking of subordinate phase.
3.3 subordinate phase the results
In first stage checking, still significant 7 SNPs enter subordinate phase checking (comprising the independent sample of 943 routine cases and 1838 example contrasts), and we find that rs12522693, rs10035791, rs80007597, rs17836917 obtain effectively verifying further with associating of colorectal cancer.In order to verify whether these sites are independently to the risk association effect of colorectal cancer, we carry out Meta-analysis combined analysis to the result of primary dcreening operation and two Qualify Phases, as shown in table 3, no matter be primary dcreening operation stage, Qualify Phase or amalgamation result, there are two site (rs12522693at5q23.3, OR=1.32, P=1.34 × 10
-8; Rs17836917at17q12, OR=0.75, P=3.62 × 10
-8) reach the significance of full-length genome.
Table 3GWAS primary dcreening operation and 4 SNPs all consistent in two phase authentication
ahigh-frequency/low-frequency allelotrope,
bhigh frequency homozygote/heterozygote/homozygote, MAF
c: less gene frequency.OR
add, P
add: be added during genetic model calculates and have adjusted age, sex and first principal component.
Embodiment 2SequenomMassArray detects the genotype in rs12522693 or rs17836917 site
(1) genome method of sample is extracted with embodiment 1.
(2) amplification comprises the region of DNA territory of target site, the primer sequence used is as follows: for rs12522693 site, amplimer sequence is: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.
(3) use shrimp alkaline phosphotase SAP remove system middle reaches from dNTP, purified pcr product;
(4) single base extension: the PCR primer after SAP process carries out the extension of base specific, there is difference in not homoallelic extension products molecular weight, namely the difference of extension products molecular weight embodies the different bases of pleomorphism site.For rs12522693 site, extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, extension primer sequence is 5 '-AATTCTGCTTCTGGAGAT-3 '.
(5) resin purification: extension product adopts resin to carry out purifying and desalts;
(6) purified product is transferred on 384 hole SpectroChip, carries out MALDI-TOF mass spectroscopy, and TyperV4.0 software output detections result, determines gene type.
If rs12522693 site allelotrope is G, namely genotype is GG, then this individuality is that colorectal cancer susceptibility is individual, if rs17836917 site allelotrope is A, namely genotype is AA, then this individuality is that colorectal cancer susceptibility is individual.
Embodiment 3 direct sequencing detects the genotype in rs12522693 or rs17836917 site
(1) genome method of sample is extracted with embodiment 1.
(2) pcr amplification a, pcr amplification (can adopt the TakaraPCRThermalCyclerPCR amplification instrument of such as Japanese TaKaRa company) is carried out to sample DNA.The primer sequence used is as follows: for rs12522693 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.Adopt 50 μ lPCR reaction systems to carry out the gene amplification of rs12522693 or rs17836917, include 1 × PCR damping fluid, 1.5mMMgCl
2, the genomic dna of 100-150ng, upstream and downstream primer is 0.5 μM, and dNTP is the ExTaqDNA polysaccharase 1.5U of 0.2mM, TaKaRa company.Pcr amplification loop parameter is as follows: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 64 DEG C of annealing 30s, and 72 DEG C extend 30s, extend 5min again after 30 circulations.Obtain the gene amplification product of rs12522693 or rs17836917.B, get 50 μ lPCR amplified productions and carry out agarose gel electrophoresis separation, blade cuts object band.Reclaim test kit (Omega company) according to E.Z.N.A. gel and require that the DNA carrying out object band reclaims purifying.Get 2 μ l recovery product UV2000 UV detector (Japanese Shimadzu Corporation) and measure ultraviolet absorption value, carry out DNA concentration determination, and ultimate density is adjusted to about 10-50ng/ μ l.
(3) product after with recovery is checked order for template, the downstream primer used during to increase is as sequencing primer, require that carrying out order-checking PCR reacts according to CEQTMDTCS-QuickStartKit sequencing kit (Beckman company of the U.S.), reaction terminates and after purifying, carries out being separated the sequence with interpretation amplified production with the CEQ8000 type gene sequencer of Beckman company of the U.S..The sequence recorded and rs12522693 or rs17836917 sequence are compared, judges wherein whether there is G or A allelotrope.If rs12522693 site allelotrope is G, namely genotype is GG, then this individuality is that colorectal cancer susceptibility is individual, if rs17836917 site allelotrope is A, namely genotype is AA, then this individuality is that colorectal cancer susceptibility is individual.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (3)
1. for detecting the purposes of the genotypic reagent of SNPmarker in the test kit preparing Colorectal Cancer Diagnosis susceptibility, it is characterized in that, described test kit comprises the genotypic reagent of detection SNPmarker; Described SNP site is rs12522693 and/or rs17836917; The genotype in described rs12522693 site is GG, represents susceptibility gene of colorectal cancer type; The genotype in described rs17836917 site is AA, represents susceptibility gene of colorectal cancer type.
2. purposes according to claim 1, is characterized in that, described reagent comprises the reagent using SequenomMassArray to detect the reagent needed for SNP site genotype and/or use needed for direct sequencing detection SNP site genotype.
3. purposes according to claim 2, it is characterized in that, the described use SequenomMassArray reagent detected needed for SNP site genotype comprises the extension primer of primer for the nucleotide sequence of the SNP site that increases and/or the reaction of single base amplification; For rs12522693 site, described amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 ', described extension primer sequence is 5 '-CCACTAGCAGTCTCAC-3 '; For rs17836917 site, described amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 ', described extension primer is 5 '-AATTCTGCTTCTGGAGAT-3 '; The described direct sequencing reagent detected needed for SNP site genotype comprises the primer of the nucleotide sequence for the SNP site that increases; For rs12522693 site, described amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCAAGATGACTCTAACTACC-3 ', reverse primer 5 '-ACGTTGGATGCCTATAAGTGCAAAGGAGAG-3 '; For rs17836917 site, described amplimer sequence is as follows: forward primer 5 '-ACGTTGGATGCCCAGCCTGGTTTGTATTGT-3 ', reverse primer 5 '-ACGTTGGATGTCCTGCCTTCTTAATTCTGC-3 '.
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| CN104711368A (en) * | 2015-04-13 | 2015-06-17 | 玉峰惠仁生物医药科技（北京）有限公司 | Noninvasive polygenic hereditary colorectal cancer detection kit based on next-generation sequencing technology |
| CN105274222B (en) * | 2015-10-19 | 2018-12-07 | 南京亿科人群健康研究院有限公司 | One kind SNP marker relevant to non-steroidal anti-inflammatory drugs prevention colorectal cancer and its application |
| CN107254531B (en) * | 2017-06-28 | 2020-09-08 | 南京医科大学 | Genetic biomarker for auxiliary diagnosis of early colorectal cancer and application thereof |
| CN111057769A (en) * | 2020-01-20 | 2020-04-24 | 广东医科大学 | Kit for detecting miR-218 gene locus related to colorectal cancer susceptibility and application method thereof |
| CN113416781B (en) * | 2021-06-03 | 2022-07-22 | 杭州医学院 | Colorectal cancer diagnosis marker and application thereof |
| CN113416782A (en) * | 2021-06-11 | 2021-09-21 | 中国科学院北京基因组研究所(国家生物信息中心) | Tumor-related SNP site primer composition and application |
| CN114107505A (en) * | 2021-12-01 | 2022-03-01 | 求臻医学科技（北京）有限公司 | Primer, detection method and kit for colorectal cancer susceptibility gene SNP detection |
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