CN102277417A - APC (Adenomatous Polyposis Coli) gene and application of singlenucleotide polymorphism thereof - Google Patents
APC (Adenomatous Polyposis Coli) gene and application of singlenucleotide polymorphism thereof Download PDFInfo
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Abstract
The invention provides a method for detecting a disease of a patient. The method comprises the following steps of: (a) recognizing a gene for displaying the disease or inhibiting the disease, or recognizing one or more singlenucleotide polymorphisms in the gene; and (b) detecting the existence or deletion of the gene or one or more singlenucleotide polymorphisms in the gene.
Description
Technical field
The present invention relates to a kind of dna sequencing technology, especially a kind of technology that detects the single nucleotide polymorphism of apc gene and other gene.
Background technology
In western countries and China, colorectal cancer is a kind of common malignancy at present.Adenomatous polyposis coli (adenomatous polyposis coli, hereinafter to be referred as APC) transgenation is first at familial adenomatous polyposis disease (familial adenomatous polyposis, FAP) find on the locus, and think that this gene is relevant with the generation of colorectal carcinoma.APC is tumor suppressor gene (seeing United States Patent (USP) RE36,713).Genome and epigenetic acting in conjunction cause the apc gene afunction, and this is bringing into play important effect in the generating process of colorectal carcinoma.Apc gene has a plurality of structural domains, can comprise β-chain of rings element, axle albumen, CtBP, Asefs, IQGAP1, EB1 and microtubule in conjunction with different albumen.United States Patent (USP) 5,998 has disclosed the APC tumor suppressor protein for No. 600 and has played important effect with the plain inhibition effect that combines the APC tumour of β-chain of rings, and APC or β-a chain of plain sudden change can destroy above-mentioned regulation mechanism.By forming the degraded mixture of being made up of axle albumen, GSK-3b and casein kinase, APC can regulate WNT/ β-a chain of plain signal transduction pathway.Many dissimilar sudden changes can cause that the function in above-mentioned key function territory loses.United States Patent (USP) 6, having disclosed nonsense mutation 013, No. 461 may be relevant with colorectal cancer, has many examples to comprise that sudden change influences key amino acid in the binding domains, form truncated protein at no main structural domain place, disturb the exon shearing in coding primary structure territory etc.In addition, APC is also bringing into play important effect in other many elementary cell activities, comprises that cell adhesion and migration, formation Actin muscle and microtubule network, spindle body form and chromosome segregation.The suddenly change imbalance of caused above-mentioned cellular activity of APC points out it may be relevant with the generation of colorectal cancer.Wood and colleague have carried out order-checking and found that apc gene is a kind of modal mutator gene (L.D.Wood etc. in colorectal cancers the genetic transcription thing from many knot rectum patients, Science, 2007,318,1108-1113), but their result all come from the exon sequence of conventional pcr amplification gene measured.
Single nucleotide polymorphism (single-nucleotide polymorphism, hereinafter to be referred as SNP) at first propose by Steve doctor Ligget, it is a kind of performance of mutant dna sequence, promptly there is single Nucleotide-A in the Different Individual (or two karyomit(e)s of same individuality) same race in genome (or other sequence), T, the difference of C or G.For instance, measure among sequence A AGCCTA and the AAGCTTA from two dna fragmentations of Different Individual and to include single different IPs thuja acid, we just have been referred to as two allelotrope: C and T so.All common single nucleotide polymorphism sequences nearly all only contain two allelotrope.See http://en.wikipedia.org/wiki/Single-nucleotide_polymorphism or other wide coverages about SNP.
In the crowd, the single nucleotide polymorphism sequence is considered to equipotential gene rate a kind of time.The gene frequency of also having observed transposon in a certain specific crowd is minimum.Two gene frequencies are simple and little with respect to single nucleotide polymorphism, but then have a lot of variations when in the crowd, occurring, thus the SNP allelotrope that occurs among a slice regional distribution or the ethnic population seldom with another sheet regional distribution or ethnic population in identical.Development along with the new-generation sequencing technology, people have reached oncogene group sequencing again and have measured, two mains direction of studying are wherein arranged: the mainly logical sudden change that detects the amplified production of full genome exon of the many technology of the first, carry out sequencing afterwards again, finished the full genome exon sequence of many tumours recently and measured, comprise mammary cancer, colorectal cancer (L.D.Wood etc., Science, 2007,318,1108-1113), carcinoma of the pancreas (S.Jones etc., Science, 2008,321,1801-1806) and glioblastoma (D.W.Parsons etc., Science, 2008,321,1807-1812); Another method is that the oncogene whole genome sequence is measured.The example that first is in the news be the research of being done by Ley etc. and Mardis etc., they adopt the Illumina sequencing technologies that new genome of former, the normal acute myeloblastic leukemia undifferentiated type of cytogenetics (AML-M1) patient and the normal skin genome that is complementary with it are checked order, 52 individual cells point mutation (T.J.Ley etc. conservative or regulator site in 12 kinds of secondary (somatocyte) sudden change that has detected gene coded sequence and the genome, Nature, 2008,456,66-72; And E.R.Mardis E.R. etc., N.Engl.J.Med., 2009,361,1058-1066.).Afterwards, people such as Shah are based on the Illumina sequencing technologies, to the primary tumo(u)r patient reached at that time this patient after 9 years metastatic lesion carry out finding behind the gene order-checking, the sudden change of 32 individual cells is arranged in metastasis, and only detected 11 (S.P.Shah etc., Nature, 2009 in primary tumor site, 461,809-813; M.J.Clark etc., PLoS Genet., 6,2010, e1000832).
Summary of the invention
From an aspect, the invention provides a kind of method that detects organism disease, its method may further comprise the steps: (a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene; (b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
From another aspect, the invention provides a kind of method of dlinial prediction disease, its method may further comprise the steps: (a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene; (b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
Still from another point of view, the invention provides a kind of method that body suffers from certain disease risks that is used for assessing, this method may further comprise the steps: (a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene; (b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
In examples more of the present invention, detect this disease of gene representation and be in holddown.
In examples more of the present invention, detected gene is an apc gene.
In examples more of the present invention, this disease is relevant with the apc gene imbalance.
In examples more of the present invention, this disease is colorectal cancer (colorectal cancer).
In examples more of the present invention, step (a) comprises the detection of one or more mononucleotide sequence polymorphisms in apc gene or the apc gene.
For example, step (a) can be finished by the method that comprises following several steps: (i) obtain apc gene full-length gene group zone, the upstream sequence of the apc gene that (ii) increases, exon, intron and downstream sequence; (iii) on the fragment matrix of apc gene, obtain specific DNA fragments; (iv) the dna fragmentation that obtains is checked order fast; (v) detect the dna sequence dna single nucleotide polymorphism of obtaining.
The alternatives of step (a) can be following wherein a kind of: allele specific oligonucleotide oligonucleotide hybridization technology, allele specific pcr, solid phase are supported micrometering preface technology, oligonucleotide to connect and are measured skill and technique, 5 FU 5 fluorouracil nuclease assay method or restriction fragment length polymorphism.
In examples more of the present invention, (Biosystems, Warrington UK) finish to measure locus (being commonly referred to the Tagman analytical method) employing AB7700 instrument by the specific type PCR that detects polymorphism.This method need be bited into probe, it can be hybridized with the fragment interested that contains polymorphism, and probe is made up of three parts: a fluorescent receptor molecule, fluorescent quenching agent molecule and energy and chemical substance bonded ditch strengthen and the combining of genomic dna chain.Probe can be incorporated on any chain of dna double chain.For example, when being attached to coding strand, when the Taq polysaccharase since 5 ' upstream promoter district synthetic DNA, can remove base on the probe one by one by 5 '-3 ' exonuclease activity if polysaccharase just runs into behind the probe.After the base of fluorescence molecule mark was removed, fluorescence molecule therefore will be luminous no longer by the quencher cancellation, and this reaction only just can take place after probe specificity ground and the hybridization of its complementary genome sequence.After continuous amplification, can be attached on the DNA in the reaction system if any more probe and primer, fluorescence is enhanced, and therefore can detect positive findings.If genomic dna does not comprise and probe complementary sequence, just detect less than fluorescent signal.PCT patent application WO 05/008555A1 being described in more detail relevant for this method.
In other example of the present invention, step (b) comprises existence or the disappearance that detects one or more single nucleotide polymorphism in this gene or the gene.
The present invention also provides a kind of method of the medicine likely effectiveness that is used for the assess patient disease treatment.This method comprises following a few step: (a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene; (b) use before this medicine, detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene; (c) use this medicine in this patient (promptly treating this patient); (d) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene after the treatment or this gene; (e) relatively treat the existence of one or more single nucleotide polymorphism in this gene of front and back or this gene or the situation of disappearance.
In some examples of this method, this gene is an apc gene.In the other example of this method, this disease is meant colorectal cancer.
The present invention also provides the adjusting apc gene active method from the another one aspect, is included in and introduces one or more single nucleotide polymorphism in the apc gene.For example, can introduce one or more single nucleotide polymorphism at apc gene promoter region, exon, intron or intron and exon junction.
In addition, range of application of the present invention also comprises the material that uses or produce in the aforesaid method.
Term used herein " individuality " is meant ill or anosis Mammals, can be people or the animal such as dog or cat.
Term used herein " detection " be meant the support of rational science or medical science or evidence diagnosis, supposition or prediction.
Term used herein " therapeutant " is meant the material that certain disease is had result of treatment.This material can be the chemistry or biological composite or constituent or mixture.
Term used herein " disease " is meant that a body suffers from pathological state tumour or that do not suffer from tumour.Tumour (can be " malignant neoplasm ") is a class because the growth of a group cell (unlimited division) out of hand, soak into (invading or destroy adjacent tissue) and sometimes to the disease of distant metastasis (being diffused into other position of health by lymph or blood pressure).The colorectal cancer that comprises, the rectum cancer, kidney, liver cancer, carcinoma of the pancreas or other tumor types that these tumours can be among the present invention to be mentioned.
Term used herein " inhibition " is meant severity or the occurrence frequency that palliates a disease, or eliminates this disease.
Description of drawings
Fig. 1 has shown concentration and the distribution of each SNP of apc gene of colorectal cancer.The concentration of the corresponding SNP sequence of the high expression of each post in the histogram, X-axis is represented APC reference position (comprising the 10k upstream of beginning and last 5k downstream).Red area is the colorectal cancer of APC, and green area is represented non-coding region.Density bullet be conservative transcription factor binding site point, blue markings be meant the miRNA target site.
Fig. 2 shows is to have confirmed that by AS-PCR SNP is positioned at No. 5 karyomit(e) 112074356 (C＞T), there is T allele-specific PCR product to occur in 16 samples, have C allelotrope in 8 samples, show that 8 samples are C/T heterozygotes, other 8 is T allelotrope homozygote.
Fig. 3 is to the SNP sequence that adopts colorectal cancer that long range PCR or NimbleGen sequence capturing technology for detection arrive and adjacent tissue relatively.SNP sequencing in the PCR-C:PCR amplification colorectal cancer sample; PCR-N: SNP sequencing in the normal adjacent tissue sample; NG-C, NimbleGen catch SNP sequencing NG-N:NimbleGen in the colorectal cancer sample and catch SNP sequencing in the healthy tissues sample.
Fig. 4 has shown the sequence distribution plan of gene sequencing technology for detection apc gene of new generation, wherein red line is represented the sequential detection concentration of relevant base, green line is represented GC content in the 100bp scope, and blue piece is represented the low-complexity zone by cross-matched distribution identification.PCR_C: the long range PCR of cancerous tissue; PCR_N: the long range PCR of normal adjacent tissue; The cancerous tissue dna sequence dna is caught in the NG_C:NimbleGen order-checking; The dna sequence dna of normal adjacent tissue is caught in the NG_N:NimbleGen order-checking.
Embodiment
The invention provides a kind of complete genomic target amplification or capture technique that comprises upstream sequence, exon, intron and downstream sequence, then by the SNP sequence of new-generation sequencing technology in effective detection great amount of samples.Apc gene is studied in great detail by people because it plays important effect in the generation of colorectal cancer and checks order, and we are the present invention of example detailed explanation with this gene just.
The present invention has at present used the target amplification technique to obtain the apc gene whole genome sequence that comprises upstream sequence, exon, intron and downstream sequence, need synthetic special primer right simultaneously, its quantity does not wait at 10～16 pairs, and is comparatively commonly used as 12～14 pairs of design of primers.Because the apc gene sequence is known at present, the synthetic of primer can be used existing mature technology.Comprise upstream 10k sequence and downstream 5k sequence area with reference to human apc gene (Ensembl Gene IDENSG00000134982), primer (S.Rozen and H.Skaletsky, Methods Mol.Biol., 2000 of adopting Primer 3 systems to design, 132,365-386).
Amplification can be adopted existing proven technique.One of them method is exactly 25 μ L system PCR, comprises 1 * long range PCR damping fluid, 2.5mM Mg
2+, each dNTP500 μ M, each primer 0.4 μ M, 1 unit long range PCR enzyme mixture (QIAGEN Inc., California, USA Valencia city).
PCR is reflected in the following thermal cycle reaction and carries out: initial denaturation temperature 93 ℃ * 3 minutes; Per 15 seconds is a circulation under 93 ℃ of conditions, carries out 10 circulating reactions altogether; Annealing temperature (seeing the following form 1) 30 seconds, 68 ℃ continue 11 minutes down; Per 15 seconds is a circulation under 93 ℃, totally 28 circulating reactions; It is a circulation that annealing temperature is descended per 30 seconds for 68 ℃, and totally 11 minutes, each additional circulation prolonged 20 seconds; Reaction is 10 minutes under 68 ℃ of final elongating temperatures.
Behind the amplified reaction, the PCR product can carry out purifying through existing mature technology.For instance, the PCR product that specific fragment has been cut can adopt QIAquick PCR purification kit and QIAquick DNA to reclaim test kit (QIAGEN Inc.) it is reclaimed purifying through after the electrophoretic separation.
Also can adopt present known technology to carry out determined dna sequence and amplification.Such as adopting conventional 385k tiling sequence capturing matrix to carry out target APC sequencing (No. 5 karyomit(e)s, corresponding 112091483-112214834bp), the sequencing capture technique of employing Roche NimbleGen obtains this and catches matrix (seeing http://www.nimblegen.com/products/seqcap/).The another one method is to adopt the SureSelect target selective system of Agilent.For detecting SNP, we collect from 30 patients' genomic dna, comprising colorectal carcinoma patient, other tumor types patient or suffer from the patient of other disease, all DNA mixed and cut into fragment, again with fragment and the hybridization of sequence capturing matrix preface, the fragment that flush away is not hybridized, last wash-out and amplified target are to the enrichment DNA sequence.
Can also adopt other known technology to come DNA amplification, 10 minutes (130w of dna fragmentation such as the supersound process amplification, Cole-Parmer CPX 130, U.S. Yi Linuo state) making it form mean size is the dna fragmentation of 500 base pairs, adopts Qiaquick PCR purifying column spinner (Qiagen Inc) to be further purified and to concentrate then.Use the T4DNA polysaccharase, the terminal genomic DNA fragment of repairing of Klenow archaeal dna polymerase and T4TNK mixture (Promega, Wisconsin, USA Madison city).The Klenow exonuclease adds (Promega) poly A tract structure 3 ', according to the Illumina specification sheets synthetic fragment is added the Illumina joint at its two ends under DNA T4 ligase enzyme (Promega) effect then, separate the dna fragmentation that is connected with joint with agarose gel electrophoresis more afterwards.Further handle the suitable big or small fragment between the 150-200 base pair, reclaim test kit (Qiagen Inc.) with QIAquick glue and cut the recovery dna fragmentation from sepharose, the Illumina joint makes the dna fragmentation amplification of cutting through 18 circulations then.According to the Illumina specification sheets, be purified with the segmental examining order of identification of dna and can after 36 circulations, finish.Alternatives: comprise ABI by sequencing equipment of new generation, the SOLID system also can carry out sequencing, and the order-checking circulation can be arbitrary cycle number (common＞18 circulations, can increase to a hundreds of circulation when technical licensing).
Adopt present known technology to detect SNP, can separate and film imaging short sequence fragment as Firecrest and Bustard system by Illumina, use BWA (0.5.3 version) then, under the condition of parameter acquiescence, record (ENSG00000134982 comprises 10kb upstream and 5kb downstream area) in the human APC reference gene group sequence.BWA is an effective system that comprises relatively short sequence, does not allow that wrong long canonical sequence of joining with the space exists, thus can find SNP sequence and deletion polymorphism (H.Li etc., Bioinformatics, 2009,25,1754-1760).Use Samtools (0.1.6 version) can detect SNP sequence and deletion polymorphism, this system can move and improve the method that its downstream data is handled, as index, overlapping, browser with share and be in harmonious proportion.Samtools can produce a shared sequence jointly with the data model that is implanted among the MAQ, and possible PCR repeats and can remove according to " Samtools rmdupse " order.Original variation may be called as " Samtools pileup " order under system default parameter situation, filtered by " Samtools.pl varFilter " order down by system default then, but except following situation: minimum readable length (5 byte) is to go to the iteron, and maximum readable length (255 byte) has also been removed the multiple problem of random permutation.Depend on the different SNP scorings of SNP identification selection, for obtaining high quality SNP, the SNP scoring is acceptables greater than 45.
May be because the obtaining of invalid sequence in the experiment, or incorrect short-movie section location, alternative approach is applicable to that those exist and do not have or low the reading covers section.GC content and readable coverage area may can calculate relation conefficient thus in one 100 base pair scope.In the scope of 100 base pairs, also can calculate the average effective scope that the PCR tumor-necrosis factor glycoproteins is removed front and back, may find repetition and low complex region by cross match system and system default setting.
Be prediction SNP sequence impact effect, the SNP sequence of removal need compare in Ensembl makes a variation database.Use SNPnexus that the SNP sequence is carried out functional annotation, comprise the impact effect of gene and controlling element.Adopt present proven technique,, or be used for RESCUE-ESE and the PAS-ESS system that ESE predicts, may need different instruments to integrate institute and predict the outcome, comprise the use of online FASTSNP instrument as ESEfinder.
For predicting whether SNP influences the combination of transcription factor, and we need use other analysis tool, as PROMO, this is a system that detects transcriptional regulatory element by use species dedicated search.
Use gene sequencing technology for detection SNP sequence of new generation, may need to use allele-specific PCR, may need to use the anti-abruptly-changing system of four primer amplifications for instance.
Below describe the present invention in detail with several examples.All be incorporated among the Ben Wenben at all reference (comprising web page contents) that this quotes in the mode of quoted passage.
Example 1: method
From the tissue sample of 6 healthy normal individuals and other 37 colorectal carcinoma patients in the control group, all individualities are the foreign citizen of Chinese origin.Institutional review board approval is obtained in collection of specimens, adopts zooblast tissue, and blood etc. are DNA purification kit (QIAGEN Inc.) and according to its specification sheets DNA isolation always.
Use the target amplification technique to obtain the full genomic gene of APC, comprise upstream sequence, exon, intron and downstream sequence.For example, when the genome of amplification apc gene, according to the upstream 10k sequence and the downstream 5k sequence area of human apc gene, use 14 pairs of Auele Specific Primers of Primer 3 system designs (S.Rozenand H.Skaletsky, Methods Mol.Biol., 2000,132,365-386).
DNA cloning adopts 25 μ LPCR reaction systems, and mixture comprises 1 * long range PCR damping fluid, 2.5mM Mg
2+, every kind of dNTP 500 μ M, every kind of primer 0.4 μ M, 1 unit long range PCR enzyme (QIAGEN Inc.).PCR reaction is according to following thermal circulation parameters: 93 ℃ of initial sex change totally 3 minutes; Per 15 seconds is a circulation under 93 ℃, totally 10 circulating reactions; 68 ℃ each circulated totally 11 minutes lasting 30 seconds to annealing temperature down; 93 ℃ per 15 seconds be a circulation, through 28 circulating reactions; It is a circulation that annealing temperature is descended per 30 seconds for 68 ℃, and totally 11 minutes, each additional circulation prolonged 20 seconds; Reaction is 10 minutes under 68 ℃ of final elongating temperatures.Behind amplified reaction, the PCR product that specific fragment has been cut can adopt QIAquick PCR purification kit and QIAquick DNA to reclaim test kit (QIAGEN Inc.) through after the electrophoretic separation.
Employing is caught with the DNA amplification sequence by Roche NimbleGen design and the 385k tiling sequence capturing matrix made, carry out target APC sequencing (No. 5 karyomit(e)s simultaneously, corresponding 112091483-112214834 base pair) from 30 colorectal carcinoma patients' genomic dna in 1: 1 ratio composition mixture and to be transported to Roche NimbleGen. brief, the genomic dna sample is cut into a certain size fragment, with conventional NimbleGen sequence capturing hybridization array, flush away is hybridized fragment not, DNA (the T.J.ALBERT etc. of wash-out and amplification probe mark, Nat.Methods, 2007,4,903-905; M.D ' Ascenzo etc., Mammalian Genome, 2009,20 (7), 424-436; M.Droege etc., J.Biotechnol., 2008,136,3-10; And D.T.Okou, Nat.Methods, 2007,4,907-909).
There are other many proven technique to be used to measure the method (as the SNP sequence) of nucleotide difference, comprise following method but be not limited to this: allele specific oligonucleotide hybridization (ASO) (R.B.Wallace etc., Nucleic Acids Research, 1981,9,879-894; S.Ikuta etc., Nucleic Acids Research, 1987,15,797-811; D.A.Nickerson etc., Proc.Nat ' l.Acad.Sci.USA, 1990,87,8923-8927; M.Verlaan-de Vries etc., Gene, 1986,50,313-320; R.K.Saiki etc., Proc.Nat ' l.Acad.Sci.USA, 1989,86,6230-6234; Y.Zhang etc., Nucleic AcidsResearch, 1991,19,3929-3933); Allele-specific PCR (C.R.Newton etc., Nucleic Acids Research, 1989,17,2503-2516; R.A.Gibbs etc., Nucleic AcidsResearch, 1989,17,2437-2448); Solid phase support micrometering preface technology (A.C.Syvanen etc., Am.J.Human Genetics, 1993,52,46-59); Oligonucleotide connects measures skill and technique (OVA) (D.Y.Wu etc., Genomics, 1989,4,560-569; F.Barany, Proc.Nat ' l.Acad.Sci.USA, 1991,88,189-193; K.Abravaya etc., Nucleic Acids Research, 1995,23,675-682); 5 FU 5 fluorouracil nuclease assay method (Holland, 1991 ﹠amp; 1992, E.Lee etc., J.Toxicologicai Society, 1998,23,140-142; U.S. Patent number: 4,683,202,4,683,195,5,723,591 and 5,801,155); Restriction fragment length polymorphism (BEEP) (H.Donis-Keller etc., Cell, 1987,51,319-337).
When the DNA amplification sequence was surveyed, it was the dna fragmentation of 500 base pairs that supersound process 10 minutes (130w, Cole-Parmer CPX130) forms mean size, and is further purified and concentrates by Qiaquick PCR purifying column spinner (QIAGEN Inc.).Repair genomic DNA fragment is terminal by the mixture that T4DNA polysaccharase, Klenow DNA and T4PNK (Promega) form, the Klenow exonuclease adds (Promega) poly A tract structure 3 ', under DNA T4 ligase enzyme (Promega) effect, add the Illumina joint according to Illumina specification sheets synthetic fragment, separate the dna fragmentation of jointing afterwards with agarose gel electrophoresis at its two ends.Further be processed into the suitable big or small fragment between the 150-200 base pair, reclaim test kit (Qiagen Inc.) with QIAquick glue and from sepharose, cut the recovery dna fragmentation, the Illumina joint makes the dna fragmentation amplification of cutting through 18 circulations then.According to the Illumina specification sheets, be purified with the identification of dna fragment its examining order and after 36 circulations, finish.
Firecrest by Illumina can separate and film imaging short sequence fragment with the Bustard system, use BWA (0.5.3 version) then, under the condition of parameter acquiescence, record (ENSG00000134982 comprises 10kb upstream and 5kb downstream area) in the human APC reference gene group sequence.BWA is an effective system that comprises relatively short sequence, does not allow that wrong long canonical sequence of joining with the space exists, thus can find SNP sequence and deletion polymorphism (H.Li etc., Bioinformatics, 2009,25,1754-1760).
Use Samtools (0.1.6 version) can detect SNP sequence and deletion polymorphism, this system can move and improve the method that its downstream data is handled, as index, overlapping, browser with share and be in harmonious proportion.Samtools can produce a shared sequence jointly with the data model that is implanted among the MAQ, and possible PCR repeats and can remove according to " Samtools rmdupse " order.Original variation may be called as " Samtools pileup " order under system default parameter situation, filtered by " Samtools.pl varFilter " order down by system default then, but except following situation: minimum readable length (5 byte) is to go to the iteron, and maximum readable length (255 byte) has also been removed the multiple problem of random permutation.The SNP scoring is considered to high-quality greater than 45.
Because obtaining of invalid sequence, or incorrect short-movie section location may cause forming nothing or low the reading covers section.GC content may influence identification of Solexa base and pcr amplification, and the low-complexity zone may influence short-movie section location.GC content and readable coverage area use R (www.r-project.org) can calculate relation conefficient (Spearman method) in one 100 base pair scope.In the scope of 100 base pairs, also can calculate the average effective scope that the PCR tumor-necrosis factor glycoproteins is removed front and back, may find repetition and low-complexity zone by cross match system (Phrap package) and system default setting.When having similarity with other sequence of finding in human genome, these low-complexity zones can cause location difficulty at that time.
Be prediction SNP functional nucleotide sequence impact effect, the SNP sequence of removal need compare in Ensembl makes a variation database (the 55th edition).Use online SNPnexus that the SNP sequence is carried out functional annotation, this is a database that the complete functional annotation of SNP sequence is provided, comprise gene and controlling element impact effect (Chelala etc., Bioinformatics, 2009,25,655-661).The function effect effect of the SNP sequence of these montages can be passed through ESEfinder system (L.Cartegni etc., Nucleic Acids Res., 2003,31,3568-3571), or be used for the RESCUE-ESE (W.G.Fairbrother etc. that ESE predicts, Science, 2002,297,1007-1013) and (Wang etc. of PAS-ESS system, Cell, 2004,119,831-845) predict.Predict the outcome may need to use online FASTSNP (H.Y.Yuan etc., Nucleic AcidsRes., 2006,34, W635-641) instrument is integrated it, because FASTSNP can be connected to different SNP function prediction systems by central server.
We adopt species dedicated search in the PROMO system (D.Farre etc., Nucleic Acids Res., 2003,31,3651-3653; X.Messeguer etc., Bioinformatics, 2002,18,333-334) detect known transcriptional regulatory element, whether prediction SNP sequence influences the combination of transcription factor.The input any side dna sequence dna of SNP sequence 10 Nucleotide are selected human species transcription factor and are selected the transcription factor site of all species.
Apc gene SNP sequence is formed.For 24 The Heterogeneity, use allele specific pcr (AS-PCR) (Wangkumhang etc., BMC Genomics, 2007,8,275 to (48 samples) colorectal cancer patients and 6 healthy volunteer's samples; S.Ye etc., Nucleic Acids Res., 2001,29, E88-88) confirm the SNP sequence of new-generation sequencing technology to measuring.The anti-abruptly-changing system of four primer amplifications (ARMS)-PCR is improved by people such as Ye, can effectively measure the gene phenotype single nucleotide polymorphism.In brief, can use the synthetic four primer ARMS-PCR of online design of primers system (http://cedar.genetics.soton.ac.uk/public_html/primer1.html) design.Each PCR is reflected in the 10 μ l reaction systems and carries out, and this system comprises 1 * TAKARAEx Taq damping fluid, 2.5mM Mg2
+, every kind of dNTP 250 μ M, every kind of primer 0.2 μ M, TaKaRaEx Taq 5U/ μ L.By 2% agarose gel electrophoresis analytical results.
Example 2: the SNP sequence of 210 high conservatives in the identification apc gene
27 pairs of colorectal cancer patients and adjacent tissue's sample are carried out the full genome of APC (123kb) amplification with long range PCR in the PCR of 14 10kb sizes reaction system, comprise 10kb upstream sequence and 5kb downstream sequence.The primer that uses in the long range PCR sees the above table 1.When we successfully increase each when zone of apc gene, the coverage of long range PCR reaches 100%.We are beneficial to the new-generation sequencing technical measurement mixing with above-mentioned consistent PCR product D NA sequence from 14 of 27 colorectal cancer samples (378 PCR products altogether), and the healthy tissues sample of other 27 straight colorectal carcinomas of vicinity adopts same treatment process.Before mixing, each individual sample increased to substitute and re-use pcr amplification after sample mixes, to determine the average representativeness of 27 pairs of last measurement results of sample.This point is very important, because different genes group PCR efficient difference (data are shown) when handling, therefore before PCR sample is mixed and may cause the final representational bias of sample.
In addition, NimbleGen sequence capturing technology (Roche Diagnostics) is used to catch same genome area, is used to detect it and whether can substitutes the big long range PCR of labour intensity.The cost that NimbleGen catches each sample is very expensive, and we plan tumor specimen storehouse and normal adjacent tissue sample storehouse are used jointly, are transported to NimbleGen and carry out sequence capturing.Repetition or low-complexity sequence are not measured in NimbleGen sequence capturing technology in the zone of apc gene, need not synthesize the probe at aforementioned region.
Use BWA (0.5.3 version) (H.Li etc., Bioinformatics, 2009,25,1754-1760), under the condition of parameter acquiescence, short sequence fragment first with human apc gene reference gene group sequence consistent (ENSG00000134982), comprise 10kb upstream and 5kb downstream area.BWA is an effective system that comprises relatively short sequence, does not allow that wrong long canonical sequence of joining with the space exists, so can find SNP sequence and deletion polymorphism.Remove and regional paired tumor-necrosis factor glycoproteins more than, SNP sequence and deletion polymorphism (are seen Li etc., supra) are detected by samtools (0.1.6 version) and MAQ.After analyzing, the quality score of SNP sequence and deletion polymorphism was greater than 45 minutes the high-quality sequence that is considered to.
We have measured 210 SNP sequences in the apc gene at last, and wherein 69 is normal SNP sequence (see Table 1 and table 2), can not find correlated series in snp database.All detected SNP sequences are created a bedGraph formatted file, be convenient to be uploaded to the intron that is used to analyze on the USCS genome browser wherein.Detect 7 SNP sequences at upstream region, 11 SNP sequences are positioned at exon (table 2).Wherein 10 is named SNP sequence, and another one is unnamed SNP sequence.Detected SNP (the human genome HG19 that a new the past do not report.Chr5:112179745) (table 2).In addition, we find that also SNP is distributed in (Fig. 1) on the apc gene fifty-fifty.Fig. 1 has shown colorectal cancer patients apc gene SNP sequence concentration and distribution, the concentration of the corresponding SNP sequence of the high expression of each post is represented the concentration of related SNP sequence in the histogram, and X-axis is represented APC reference position (comprising the 10k upstream of beginning and last 5k downstream).Red area is the colorectal cancer of APC, and green area is meant non-coding region.Density bullet be conservative transcription factor binding site point, blue markings be meant the miRNA target site.But viewed these SNP function effect effects await further research.
Table 1. Chinese knot rectal cancer patient apc gene SNP sequencing is summed up
* the highest SNP scoring in four sequencing systems
* Ensembl variation database V55 note
The apc gene exon SNP sequence of all 4 mensuration of table 2.
Table 3.APC gene upstream sequence SNP sequence and their TF are in conjunction with effect
Example 3: to detecting the prediction of result function of SNP sequence
Not only obtained the SNP sequence of coding exon in order to last method, also detect the SNP sequence of upstream sequence, intron and downstream sequence simultaneously, being necessary more has comparative analysis to the function of the SNP sequence that detects, rather than simply uses coding SNP name or unnamed to its note.Human genome regulates and control to reach the purpose of carrying out correct montage in tram and orthochronous mRNA sequence by different cis-acting elements.The SNP sequence influences the controlling element in the montage process, and then influences gene splicing, has to discover that this effect has caused human numerous disease to comprise generation (L.Cartegni etc., Nat Rev Genet., 2002,3, the 285-298 of cancer; S.Wang etc., Nat Rev Genet., 2007,8,749-761).These controlling elements comprise intron and exon montage enhanser (ISE and ESE) (L.Cartegni etc., NucleicAcids Res., 2003,31,3568-3571) and suppress (ESS and ISS) element (Z.Wang etc., Cell, 2004,119,831-845).Exon montage enhanser (ESEs) is the cis acting controlling element, instruct or help the correct montage (Cartegni etc. of its precursor RNA sequence, 2003, supra), exon montage silencer (ESSs) also is the use (Wang etc. that cis-regulating element can suppress contiguous splice site, 2004, supra).Exon montage enhanser (ESEs) can with the SR protein binding, raise and stable the essential albumen of the sub-montage of montage, and exon montage supressor (ESSs) energy syncaryon unhomogeneity ribonucleoprotein (hnRNP) albumen composition further suppresses the use (Wang etc. of exon, 2007, supra).
Use online tool FASTSNP (Yuan etc., 2006, supra) predict the enhancing of ESE or ESS or lose, we find that 8 kinds have been named in the SNP sequence 7 kinds because the enhancing of ESE or ESS or lose and influence splicing (table 2) in the coding region.Two kinds in three kinds of SNP sequences of 3 ' non-coding region have also caused the enhancing of ESE/ESS or have lost (table 2).A kind of in the unnamed SNP sequence also caused ESE enhancing (table 2).It is to be noted that PolyPhen has predicted this unnamed SNP, also having predicted amino acid simultaneously replaces and may influence (http://genetics.bwh.harvard.edu/pph/) (V.Ramensky etc. to people's protein structure and function, Nucleic Acids Res., 2002,30,3894-3900), have only protein function is had optimum effect (PSIC difference scoring 0.532).Yet it has caused the increase of exon montage enhanser.
SNP sequence detected (table 1) in apc gene upstream sequence (being considered to promoter region) 7.We adopt, and the species dedicated search detects known transcriptional regulatory element (Farre etc., 2003, supra in the PROMO system; Messeguer etc., 2002, supra), whether prediction SNP sequence influences the combination of transcription factor.We find to fix on No. 5 karyomit(e): 112070456 (the SNP sequence prediction on the G-＞A) position (all is with reference to human genome HG19 position) is to there being two transcription factors combinations to lose-c-Ets-1[T00112] and R2[T00712] (table 3).Be positioned at karyomit(e) No. 5: 112067142 (the SNP sequence on the C-＞T) can cause YY1 in conjunction with increase, and the additional binding site of ENKTF-1 (from or 2 sites) increases.Be positioned at karyomit(e) No. 5: 112066524 (G-＞C) can cause that the TF binding site increases, they can with p300[T01427], R2[T00712], NFI/CTF[T00094] and EIk-1[T00250] combination.Be positioned at karyomit(e) No. 5: 112064826 (SNP of G-＞C) can cause PR B[T00696] and PR A[T01661] in conjunction with losing, c-Myb[T00137 at that time] in conjunction with increasing.Be positioned at karyomit(e) No. 5: 112063970 (SNP of G-＞C) may cause WT1I-KTS[T00900], WT1-KTS[T01839] and ETF[T00270] in conjunction with increasing (table 3).
A large amount of SNP sequences in the apc gene on the intron are detected by aforesaid method.Recent genomic data show intron comprise the important zone of many functions as conservative intron montage controlling element (ISREs) to generegulation and montage play an important role (See G.W.Yeo etc., PLoS Genet., 2007,3, e85).These ISRE sequences comprise intron montage enhanser (ISEs) and intron montage silencer (ISSs).The ISE sequence can strengthen the exon effect and activate weak exon, and the ISS sequence can suppress the exon effect and create reticent regional (M.Y.Chou etc., Mol.Cell, 2000,5, a 949-957 by raising the montage repressor; R.Singh etc., Science, 1995,268,1173-1176; J.Valcarcel etc., Nature, 1993,362,171-175).Have been found that by UCSC genome browser (data not shown) many detected SNP sequences are positioned at the intron zone of evolution conservative.Yet effect detects to ISRE region S NP sequence because there is not reliable prognoses system at present, and the functional conclusion of SNP sequence needs further checking.
Example 4: adopt allele specific pcr to determine the SNP sequence
For clear and definite our the SNP sequence that method detected is real SNP sequence, use allele-specific PCR (AS-PCR) that the SNP sequence of picked at random is measured.We adopt the method for four primer allele specific pcrs, and this is the easy and economic method of a kind of SNP genotype identification and affirmation.For increasing the allele specific pcr distinguishing ability, adopt the measure of the conventional allele specific pcr specificity method of a kind of known increase, promptly introduced the mispairing (S.Ye etc. in another 3 ' terminal-2 site, NucleicAcids Res., 2001,29, E88-88; S.Ye. etc., Nucleic Acids Res., 1992,20,1152.).
10 kinds of SNP sequences among several SNP that the difference of having selected AS-PCR to confirm is at random marked, wherein＞45,2 SNP scorings＜45 of the SNP of 8 SNP sequences scoring.24 pairs of dna sequence dnas (48 tumor specimens altogether) and 6 normal healthy controls samples from tumour and normal adjacent tissue adopt the AS-PCR amplification, all 8 SNP sequences of high SNP scoring confirm in succession, comprising a new SNP (table 4) who all exists at tumour and normal adjacent tissue.Yet SNP scoring is lower than in two SNP sequences of 45 has only confirmed one (table 4).Fig. 2 has shown by what AS-PCR determined and has been positioned at karyomit(e) No. 5: the 112074356 (SNP of C＞T).In the PCR product, T allelotrope is present in 16 samples, and C allelotrope only exists in 8 samples, shows that 8 samples are heterozygosis C/T, and other 8 samples are the T allelotrope that isozygotys.The result shows that the T allelotrope that is positioned at this position is a common allele in Chinese population.We adopt＞=45 as the sections of determining the scoring of SNP sequence, and all 8 sample SNP sequences of＞=45 all are determined, and therefore false positive rate can be ignored in 210 SNP sequences.It is in order to ensure detecting SNP sequence high quality that the scoring of SNP sequence is selected in strict control, yet we may be because some possible SNP sequences have been lost in the scoring of this strictness.
Table 4. allele specific pcr is determined the SNP sequence
Example 5 long range PCR targets are selected and new-generation sequencing technology sequence capturing technology compares
Adopt two kinds of methods that the SNP of the survey sequence distribution of tumour and normal adjacent tissue is studied, find half SNP sequence (52% approximately in four comparative group, in 210 SNP sequences 109) normal: PCR-C (pcr amplification tumor tissues specimen dna sequence), PCR-N (the normal adjacent tissue of pcr amplification specimen dna sequence), NG-C (the Nimblegen technology is caught the tumor tissues sample) and NG-N (the Nimblegen technology is caught the normal adjacent tissue sample) are (Fig. 3).In Fig. 3, PCPC represents pcr amplification colorectal cancer sample SNP sequencing; PCPC:PCR amplification normal adjacent tissue sample SNP sequencing; The NG-C:NimbleGen capture technique is to normal adjacent tissue sample SNP sequencing.
In tumour and the normal adjacent tissue sample after the comparison of SNP sequence, we find altogether (PCR-C22 of 37 SNP sequence, NG-C11) only in tumor specimen, detects (table 1), 4 function effect effects that prediction has been arranged wherein, one may cause that in 112179745 (C to A) site coding SNP sequence exon montage enhanser loses, and other 3 SNP sequences that are positioned at the apc gene upstream sequence may measurable TF bonded increase or lose (table 5).For instance, SNP 112070456 (G to A) may cause c-Ets-1[T00112] and R2[T00712] in conjunction with losing, await further to study yet whether the detected SNP sequence in most tumour intron zone relevant with colorectal cancer and how these SNP sequences work.
Use the sequence label of specific localization to detect sequence iteron on the apc gene, we find that the iteron on average it seems all similar (Fig. 4).In Fig. 4, red line is represented the sequential detection concentration of relevant base, and green line is represented GC content in the 100bp scope.Blue piece is represented the low-complexity zone by cross-matched distribution identification.As if yet at the GC enrichment region, round pcr is better than NimbleGen target capture technique, because the GC enrichment region comes mark with arrow among Fig. 4, round pcr (PCR-C group) is more than the iteron that NimbleGen capture technique (NG-C group) is measured.The low repeat region of the great majority that occur in the analysis mainly is because the label of repetition or low complex sequence has been filtered (representing with blue square among Fig. 4).
As above given example adopts the new-generation sequencing technology to genome area order-checking predetermined in the great amount of samples for finding new SNP sequence, and uses target gene amplification and capture technique to confirm.Our technology and designed lines are summarized as follows: we at first adopt length to catch the estimation range apart from genome pcr amplification or NimbleGen sequence capturing technology, adopt the SNP sequence in the new-generation sequencing technology for detection compound sample subsequently.Use then BWA (H.Li H., and R.Durbin, Bioinformatics, 2009,25,1754-1760) and samtools (H.Li etc., Bioinformatics, 2009,25,2078-2079) analytical data.In large numbers of samples, adopt allele specific pcr (AS-PCR) (Wangkumhang etc., 2007, supra; S.Ye etc., Nucleic Acids Res., 2001,29, E88-88) confirm the SNP sequence that checked order.Adopt information biology instrument SNPnexus and FastSNP (Chelala etc., 2009, supra at last; Yuan etc., 2006, supra) detected SNP sequence is carried out mark and drawn the function effect effect.
With the apc gene is example, we in the accidental colorectal cancer patients of Chinese population wherein the 27 pairs of tumours and normal adjacent tissue sample apc gene (123k) carried out genome sequencing, comprising upstream 10kb and 5kb zone, downstream.We check order by (table 1) to 210 SNP sequences altogether, and wherein 69 is newfound SNP sequence, do not report in Ensembl database (delivering 55 pieces) that the Ensembl database comprised 628 apc gene SNP sequences that detected altogether.Our research thinks that predicting genome area for one adopts target to catch or amplification technique, and using the new-generation sequencing technology subsequently is to detect the effective ways of SNP sequence in big crowd patient, and does not need the whole genome sequence is measured.Present method will be used for effective rapid evaluation influences tumorigenic important gene or genome area SNP sequence spectrum, the apc gene relevant with colorectal cancer of giving an example at this as us.Present method not only can detect exon SNP sequence, and can measure the SNP sequence in intron, upstream and downstream zone, then three zones are considered to bringing into play more and more important effect (O.Jaillon etc. in generegulation and human diseases, Nature, 2008,451,359-362).Because it is ripe that the identification bar code technology in the new-generation sequencing technology it seems that at present is that all right, present method needs further to confirm.In recent years, obtained some progress in the multiple sample of employing identification bar code joint marker and will be better than the new-generation sequencing technology, effective ways of removing the sample mixing step may be able to be provided.
In the single-shot colorectal cancer patients, adenomatous polyposis coli (adenomatous polyposis coli) (APC) gene is a kind of common sudden change in familial polyposis coli (FAP) patient.Because apc gene and FAP have been found in research, is colorectal cancer or cancer of the stomach relevant, at accidental data storehouse mutDB (http://mutdb.org/cgi-bin/mutdb.pl? id=APC﹠amp; Geneid=324) one has reported 47 kinds of nsSNP sequences (unnamed SNP) and 11 kinds of name SNP sequences in, in more detail, detected V890I in the colorectal cancer, S906Y, E911G, Y1027C, T1313A and A1508V suddenly change (M.Miyaki etc., Oncogene, 1997,15,2877-2881) (http://mutdb.org/cgi-bin/mutdb.pl? id=APC﹠amp; Geneid=324).In this research, we have detected a new SNP (table 2), have increased the sudden change number relevant with colorectal cancer.In new SNP (table 1) sequences of 69 examples some may be relevant with the Chinese population specificity, with familial polyposis coli patient apc gene in the observed different crowd at present be differentiated (T.M.Attard etc., Cancer Genet Cytogenet., 2007,172,180-182).Therefore we need adopt white people and National People's Congress's sample population comparative studies to come further clearly.
Apc gene (ENSG00000134982) comprises the albumen that 2,843 amino-acid residues of 16 exons codings are formed.APC albumen has a plurality of functional domains: oligomerization structural domain and albumen n end Armadillo repeat; The terminal Mammals homology large protein (DLG) of plain a plurality of binding sites of β-chain of rings and PROTEIN C; 7 zones of 20 amino acid multiple, 15 amino acid iterons and central positions (R.Fodde etc., Eur J Cancer, 2002,38,867-871; T.Senda etc., Med Mol Morphol., 2007,40,68-81; K.J.Smith etc., Proc.Natl.Acad.Sci.USA, 1993,90,2846-2850).Apc gene by and axle plain with the β-chain of rings connect plain combine adjusting Wnt signal transduction pathway (Smith etc., 1993, supra), blocking that the inactivation of apc gene and transgenation cause in the tumour relevant (Smith etc., 1993, supra).The apc gene afunction can not suppress the Wint signal transduction pathway, thereby causes tumor cell proliferation (I.Nishisho etc., Science, 1991,253,665-669; K.Aoki etc., J Cell Sci., 2007,120,3327-3335.).APC simultaneously also with other protein binding, activate guanine nucleotide exchange factor as APC, shock protein superfamily associated protein 3, IQGAP1, microtubule, EB1 and DLG (Aoki et al, 2007, supra).
People generally believe that exons mutation causes that pathogenic effect derives from the precognition effect to reading frame and protein function.Usually the name sudden change is considered to relevant in many changes of proteins encoded function with optimum unnamed sudden change.Yet discover that recently sudden change generation or destruction montage enhanser and silencer can cause human relative disease (L.Cartegni etc., Nat Rev Genet, 2002,3,285-298; F.Pagani etc., Nat Rev Genet, 2004,5,389-396) changed our viewpoint in the past.Ji Lei evidence shows that the relevant alternative montage incident of many tumours occurs in the sudden change disappearance of influenced gene gradually, with the development of tumour and to the reaction of treatment relevant (A.R.Grosso etc., EMBO Rep., 2008,9,1087-1093).Find that at present about 50～60% cause the sudden change of disease to influence montage process (Cartegnia etc., 2002, supra; N.Lopez-Bigas etc., FEBS Lett., 2005,579,1900-1903) for instance, Pagani etc. discover that recently about 1/4th synonyms replacement causes that the montage in cftr gene the 9th and 12 exon wide range of systems sudden changes changes, and said mutation causes cystic fibrosis (F.Pagani etc., Proc.Natl.Acad.Sci.USA, 2005,102,6368-6372).
The montage of replacing provides multi-functional means for the metazoan generegulation, and individual gene can produce multiple transcribing by different montages, has therefore enlarged the diversity of transcripton and promotor.Getting on from premessenger RNA comprises small nut ribosomal protein (snRNP) and external non-small nut ribosomal protein (non-snRNP) except that intron, the protein splice factor, and premessenger RNA is assembled and is formed transcripton.Many distinguished sequences are positioned at 5 ' and 3 ' splice site near, the mutual relationship of montage and premessenger RNA can be regulated in the intron zone, and then influences interchangeable montage.We find in the apc gene that wherein 6 kinds and unnamed sudden change of 7 kinds of name sudden changes can predict the change (table 2) of exon montage enhanser (ESE) or exon montage silencer (ESS).Exon montage enhanser (ESE) sudden change may cause prematurity mRNA to comprise relevant exon, the silencer of exon montage at that time (ESS) sudden change the having increased exon that it comprised.ESE or ESS sudden change may influence regulates proteic specific combination, as SR albumen (being rich in Serine/arginic albumen) or heterogeneous nuclear (hn) RNPs (B.R.Gravelley, RNA, 2000,6,1197-1211).Some silencers not with regulate protein binding, can form special preceding m-RNA secondary structure, hinder the contiguous montage enhanser of SR albumen identification (Buratti etc., Mol Cell Biol., 2004,24,1387-1400).ESE/ESS increases or the accurate mechanism of losing also needs further research in our the detected apc gene sudden change.This mechanism may encode core APC functional domain exon appearance or get rid of relevantly, perhaps change the ratio of different mRNA montage hypotypes or the like that influenced.
We have also detected the SNP sequence at upstream region simultaneously, and this zone is considered to influence the transcription factor binding site point (table 3) of apc gene, can also influence apc gene simultaneously and express.In these SNP sequences wherein three kinds only in tumor specimen, detect (table 5), may cause and Wilms tumour WT1 albumen [WT1I-KTS as No. 5 karyomit(e): 112063970SNPG-＞C, WT1 lacks the replacement splicing variants that 17AA (250-266SF of WT1+KTS), 408-410 (K-T-S) and WT1-KTS replace splicing variants and lack WT1 total length AA 408-410 (K-T-S)] two hypotypes in conjunction with increasing the increase of ETF transcription factor.Wilm tumour 1-KTS hypotype (WT1-KTS) can induce the apoptosis that p53 relies on (A.L.Menke etc., Cancer Res., 1997,57,1353-1363).The promoter transcription of the no TATA box of ETF specificity activation (R.Kageyana etc., J.Biol.Chem., 1989,264,15508-15514).No. 5 karyomit(e)s: 112070456SNP A-＞G may cause c-Ets-1[T00112] in conjunction with losing, C-Ets-1 is that a kind of proto-oncogene is controlled many genes and comprised expression of gene (E.S.Reddy etc., Oncogene Res., 1988 in a large amount of extracellular matrix remodeling processes, 3,239-246; And N.Takai etc., Cancer, 2000,89,2059-2067).
Table 5. is detected SNP sequence in the colorectal cancer sample only
Relatively obtain the specific gene zone by long range PCR technology and NimbleGen target capture technique, and then use the new-generation sequencing technology.As if as the GC rich region, round pcr is better than NimbleGen target capture technique.
The effective ways of finding SNP in the target gene group zone of a large amount of samples may be all useful to many different genes.The present invention catches and the new-generation sequencing technology in conjunction with long range PCR or NimbleGen order-checking, for apc gene, aforesaid method has detected in 210 SNP sequences of colorectal carcinoma patient's tumor tissues and normal contiguous tumor tissues and has found 69 new SNP sequences.
Other example
Described above and example that provide mainly is in order to describe the present invention in detail, can't to be considered to limit range of application of the present invention.Although other example that the technology of the present invention is used is not in this description, experimental technique skilled person can be easy to expect other use range of the present invention after seeing relevant information of the present invention.
Claims (22)
1. technology that detects organism disease may further comprise the steps:
(a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene;
(b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
2. the technology of detection organism disease according to claim 1 is characterized in that detecting this disease of this gene representation and is in holddown.
3. the technology of detection organism disease according to claim 2 is characterized in that disease to be detected is a colorectal cancer.
4. the technology of detection organism disease according to claim 2 is characterized in that this gene is an apc gene.
5. the technology of detection organism disease according to claim 2 is characterized in that step (a) comprises the detection of one or more mononucleotide sequence polymorphisms in apc gene or the apc gene.
6. the technology of detection organism disease according to claim 2 is characterized in that step (b) comprises existence or the disappearance that detects one or more single nucleotide polymorphism in this gene or the gene.
7. the technology of detection organism disease according to claim 6 is characterized in that step (a) comprises the detection of one or more mononucleotide sequence polymorphisms in apc gene or the apc gene.
8. the technology of detection organism disease according to claim 7 is characterized in that detected disease is a colorectal cancer.
9. the technology of detection organism disease according to claim 1 is characterized in that described step (a) is the detection to one or more mononucleotide sequence polymorphisms in the apc gene.
10. the technology of detection organism disease according to claim 9 is characterized in that step (a) comprises following step:
(i) obtain apc gene full-length gene group zone,
(ii) the increase upstream sequence of apc gene, exon, intron and downstream sequence;
(iii) on the fragment matrix of apc gene, obtain specific DNA fragments;
(iv) the dna fragmentation that obtains is checked order fast; With
(v) detect the dna sequence dna single nucleotide polymorphism of obtaining.
11. the technology of detection organism disease according to claim 9, it is characterized in that described step (a) comprises allele specific oligonucleotide oligonucleotide hybridization technology, allele specific pcr, solid phase is supported micrometering preface technology, oligonucleotide connects measures skill and technique, 5 FU 5 fluorouracil nuclease assay method or restriction fragment length polymorphism.
12. a technology that is used for certain disease of dlinial prediction comprises several several steps down:
(a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene;
(b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
13. the technology of certain disease of dlinial prediction according to claim 12 is characterized in that described gene is an apc gene.
14. the technology of certain disease of dlinial prediction according to claim 12 is characterized in that described disease is a colorectal cancer.
15. one kind is used for assessing the method that body suffers from the risk of certain disease, may further comprise the steps:
(a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene;
(b) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene.
16. assessment body according to claim 15 suffers from the method for the risk of certain disease, it is characterized in that described gene is an apc gene.
17. assessment body according to claim 15 suffers from the method for the risk of certain disease, it is characterized in that described disease is a colorectal cancer.
18. the method for a medicine likely effectiveness that is used for the assess patient disease treatment comprises following a few step:
(a) discern the gene that shows this disease or suppress this disease, or discern one or more single nucleotide polymorphism in this gene;
(b) use before this medicine, detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene or this gene;
(c) use this medicine in this patient;
(d) detect the existence or the disappearance of one or more single nucleotide polymorphism in this gene after the treatment or this gene; With
(e) relatively treat the existence of one or more single nucleotide polymorphism in this gene of front and back or this gene or the situation of disappearance.
19. method according to claim 18, its specificity are that described gene is an apc gene.
20. method according to claim 18 is characterized in that described disease is a colorectal cancer.
21. regulate the active method of apc gene for one kind, be included in and introduce one or more single nucleotide polymorphism in the apc gene.
22. method according to claim 21 is included in apc gene promoter region, exon, intron or intron and one or more single nucleotide polymorphism are introduced in the exon junction.
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