SNP 研究与应用.ppt

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1、SNP研究与应用研究与应用 汇报人：刘希华汇报人：刘希华 电话：电话：010-62889655Email：Whycanttheywalk?警察捉搶匪與致病基因定位兩個故事情節的比較1、Genetic variation and SNPs2、SNP features3、Why SNPs?SNPs effectsSNPs and diseases4、Methods and technologies in SNP research5、SNP databasesContentsThe Challenge after Human Genome ProjectUnderstanding the link

2、between DNA sequence(Genotype)A C A C G C A .T C G A G T C.A C A C G C A .T C G G G T C.100%genetic100%environmentalBiology(Phenotype)EnvironmentGenetic VariationsChromosome numbersSegmental duplications,translocations and deletionsSequence RepeatsTransposable ElementsShort deletions and insertionsT

3、andem RepeatsNucleotide Insertions and Deletions(Indels)Single Nucleotide Polymorphisms(SNPs)MutationsSizableMinorWhy does one man live to celebrate his hundredth birthday with a glass of wine in one hand and a cigar in the other while another succumbs in midlife to cancer or heart disease?And why m

4、ay one womans breast cancer be effectively eradicated while anothers shows no significant response to the same treatment?The explanations may reside in the cumulative effect of a small number of differences in DNA base sequence called single-nucleotide polymorphisms(SNPs),which underlie individual r

5、esponses to environment,disease,and medical treatments.SNPs are the most common type of sequence variation.human Genome News.Vol 10,1999“Genetics is the study of inherited variation,DNA sequence variation”定义：不同个体间，基因组中单个碱基上的变异以及小片段的插入缺失。19961996年 LanderLander首次提出，迄今已发现SNP约 900 900 万个，相关的文章 7000 7000

6、 余篇。A C G T G T C G G T C T T A A A Maternal chromosomeA C G T G T C C G T C T T A A A Paternal chromosomeA C G T G T C G G T C T T A A A Maternal chromosome A C G T G T C G G T C T T A A A Paternal chromosomeA C G T G T C C G T C T T A A A Maternal chromosomeA C G T G T C C T A C T T A A A Paternal

7、 chromosomeThe position of the SNP is indicated by the box.Individual 1 is heterozygous,while individuals 2 and 3 are homozygous.Individual 1Individual 2Individual 3SNPSingle nucleotide polymorphism(SNP)Genetic MarkersMorphological markersCytological markersBiochemical and physiological markersMolec

8、ular markers1980,RFLPs(restriction fragment length polymorphisms)1985,STRs (short tandem repeats,mini-satellites)1990s SNPs (single nucleotide polymorphisms)RFLPs(restriction fragment length polymorphisms)限限 制制 性性 片片 段段 长长 度度 多多 态态 性性1980年，年，Botstein首次提出。首次提出。1987年，第一张较完整的人基因连锁图年，第一张较完整的人基因连锁图,包含包含3

9、93个个RELP位点。位点。亨廷顿舞蹈症（亨廷顿舞蹈症（Huntington）成为第一个使用成为第一个使用RFLP定定位的遗传病。位的遗传病。缺点：数目少，多态性不够高，在染色体上分布不均缺点：数目少，多态性不够高，在染色体上分布不均匀；成本昂贵，操作繁琐，不易发展到自动化水匀；成本昂贵，操作繁琐，不易发展到自动化水平。平。酶切和电泳酶切和电泳广泛存在，占约人类基因组的广泛存在，占约人类基因组的 5%，为为2-6 bp 的的串联重复，其中以串联重复，其中以 (CA)n，(GT)n 常见。常见。1996年，建立了以年，建立了以6000多个多个 STRs 为主体的遗传为主体的遗传图谱，两个标记之间

10、的平均距离为图谱，两个标记之间的平均距离为0.7cM。优点：数目多，信息量大；易实现自动化。优点：数目多，信息量大；易实现自动化。STR位点已经有近万个，平均位点已经有近万个，平均100-200kb有一个有一个STR位点，杂合性高。位点，杂合性高。全基因组扫描的重要手段。全基因组扫描的重要手段。STRs(short tandem repeats)短串联重复序列短串联重复序列Based on nucleotides:GA,CT,GT,AC,AT,GC；The most common type:GA and CT(Tansition，转转 换换),each possesses 30 of tota

11、l，the other 4 types are transversions(颠换颠换),10 of eachBased on the location at chromosome，coding-region SNP(cSNP),perigenic SNP(pSNP),and intergenic SNP(iSNP)Based on the frequency in a population:low and high frequency SNPAssorted by the International HapMap consortium based on the level of validat

12、ion:verified、two-hit、jsnp/perlegen-verified、bac-overlap、tsc-overlap、non-validated http:www.hapmap.org/downloads/snp_categories.htm.Types of SNPsSNPs:the most common genetic variantsl1/1000:Any two individuals share 99.9%of sequence homologyl1/300:The typical frequency of SNPs in a whole population i

13、s 1/300bpl90%of variation:SNPs are the most common genetic variantslUneven distribution:SNPs are not uniformly spaced in genomel10,000,000s:Estimation of 10 millions of SNPs in human genome and several millions single base differences between any two individuals.lHow many now?9 millions in dbSNP dat

14、abaseSNP Effects（1）SNPs in genesIn coding regions(possible protein structure changes)Synonymous substitutions(同义同义)Missense substitutions(错义错义)Nonsense substitutions(终止终止)In coding and non-coding regions Change of gene expression(by diverse binding various factors)Yield Timing Alternative splicingSN

15、Ps in regulatory regionsChange of gene expressionSNPs in non-regulatory intergenic regionsCan be used as genetic markers1234121243Methods and technologies in SNP studies Discovery(Find SNPs)Validation(A common one or rare one)Genotyping(Frequency in population)Consideration:Call ratesFlexibilityThro

16、ughputCost Fundamentalapproaches large-scalesequencingbased:genomic-alignment(GA),reducedrepresentationshotgun(RRS)PCRbased:commonPCRhybridizationbased:DNAchipsGenomic DNAmRNABAC librarycDNA libraryRRS(reduced representation shot-gun)library or samplingBAC overlapShotgun overlapEST overlapSequence o

17、verlap SNP discoveryGTTTAAATAATACTGATCAGTTTAAATAATACTGATCAGTTTAAATAGTACTGATCAGTTTAAATAGTACTGATCAHow to discover SNPsThe principle of RRS restriction enzyme digestion(Bgl II,Pvu II)given size range of restriction fragments which comprise about 0.5%of the whole genome randomly sequencing clones(twofol

18、d coverage on average)Base-callingQuality determinationContig assemblySequence viewingPolymorphism taggingPolymorphism reportingIndividual genotypingPolymorphism detectionPolyPhredConsedAnalysisSequencePhredPhrapAmplify DNA53Discovering SNPs by SequencingPhylogenetic analysis ATAGACG ATACACG ATAGACG

19、 ATACACGATAGACGATACACGHomozygotesHeterozygoteQuality control of sequence to make sure the existence of SNPs rather than sequencing error How?the quality of SNP position the quality of neighboring bases the alignment condition of flanking sequence the number of SNPs in one read 早期早期SNP检测技术检测技术1.1.限制性

20、酶切技术限制性酶切技术 RFLPRFLP 2.2.识别异源双链技术识别异源双链技术uDHPLCDHPLC（变性高效液相层析）变性高效液相层析）uDGGEDGGE（变性梯度凝胶电泳）变性梯度凝胶电泳）uSSCPSSCP（单链构象多态性）单链构象多态性）uEMCEMC（错配的酶促切割法）错配的酶促切割法）uCCMCCM（错配的化学切割法）错配的化学切割法）3.3.同源杂交技术同源杂交技术 ASO(Allele Specific ASO(Allele Specific OligonucleotidesOligonucleotides)SNP检定检定 Genotyping目标：灵敏、准确、简单、高通量

21、、低成本目标：灵敏、准确、简单、高通量、低成本Invader(Third Wave)、SNPlex(ABI)、Parallele、BeadArray(Illumina)Fluorescence Polarization(PE)、MassArray(Sequenom)SNaPshot(ABI)、SNuPe(GE)、TaqMan(ABI)、PyrosequencingThroughput常用常用SNP检测技术检测技术1.基于基于PCR引物延伸引物延伸 Pyrosequencing、MassArray Fluorescence Polarization、SNuPe SnapShot2.基于探针杂交基

22、于探针杂交TaqMan、Invader、BeadArray、Parallele、SNPlex、Chip(Affymetrix)TaqMan Target 1Invader OligoTWild Type ProbeQVR1Fluorescence 1AAflapreleased 5 flapSite of CleavageFRET Probe5555533Site of Cleavage Target 2Invader OligoGMutant ProbeQVFluorescence 2CCCflapreleased 5 flapSite of CleavageAlternate Dye Pr

23、obe55555333Site of Cleavagereleased 5 flapreleased 5 flap3AR2R1R2Invader(侵染探针技术)利用FEN1酶的酶切特性Fluorescence Polarization(1)荧光偏振技术荧光偏振技术Fluorescence Polarization(2)Fluorescence Polarization(3)DNA 提取提取自动序列分析及等位基因信息的获得自动序列分析及等位基因信息的获得PrimerMassArray（1）目标序列的扩增目标序列的扩增第一次纯化和第一次纯化和SNP位点延伸反应位点延伸反应点样点样MALDI-TOF

24、 质谱测定质谱测定Allele 1Allele 1Allele 2Allele 2+Enzyme+ddATP+dCTP/dGTP/dTTPAllele 1Unlabeled Primer(23mer)T C TExtended Primer(24mer)T C TAT GAAllele 2Unlabeled Primer(23mer)A C TExtended Primer(26-mer)A C TMassArray（2）1.SNP位点信息放大位点信息放大AGAddressP3P1P2P1P2P3AGA/AAGA/GAGG/GAllele Specific ExtensionPCR with

25、common primersProduct capture by hybridization to arrayreadoutAddressBeadArray2.2.磁珠解码磁珠解码 1.一个光纤束由一个光纤束由5万条光纤组成，每一条万条光纤组成，每一条光纤由不可腐蚀的外壁和可腐蚀的内壁光纤由不可腐蚀的外壁和可腐蚀的内壁组成。组成。2.用试剂腐蚀光纤束，形成小井。每一用试剂腐蚀光纤束，形成小井。每一个小井可容纳一个磁珠。磁珠上带有一个小井可容纳一个磁珠。磁珠上带有一段段zipcode序列，与序列，与Allele specific probe 上的上的address 序列互补序列互补。3.磁珠通过

26、光纤受到激发，磁珠通过光纤受到激发，所带集团发出荧光被仪器所带集团发出荧光被仪器探头检测。探头检测。BeadArray4.高通量高精确度的秘密：高通量高精确度的秘密：5万个磁珠中含有万个磁珠中含有1500 种与种与address互补的互补的zipcode序列序列5万个磁珠中每个万个磁珠中每个zipcode序序列重复约列重复约35次次3.检测检测将解码的光纤束插入将解码的光纤束插入“1500重重”PCR扩增后扩增后的试管进行杂交，光信号的试管进行杂交，光信号经过光纤管传导，由于光经过光纤管传导，由于光线管壁不透光，避免了信线管壁不透光，避免了信号间的干扰。由计算机将号间的干扰。由计算机将光信号解

27、析信息，实现光信号解析信息，实现genotyping。BeadArrayASO1ASO-Linker 1LSOLSO-LinkerPPSNPlexAPPASO2ASO-Linker 2GPPCGPPP=Spacer 1=Spacer 2=ZipCodes=Genomic Equiv.RegionsSNPlex=Spacer 1=Spacer 2=ZipCodesBiotinGBiotinuniversal primeruniversal primerBiotinSNP 1SNP 2SNP 3SNP数据库数据库The SNP Consortium(TSC)http:/snp.cshl.orgdb

28、SNP(NIH)http:/www.ncbi.nlm.nih.gov/SNP/index.htmlHuman Genome Variation Database(Celera and Pharmacia)http:/hgvbase.cgb.ki.se/Human Gene Mutation Database(University of Wales College of Medicine)http:/archive.uwcm.ac.uk/uwcm/mg/hgmd0.htmlHuman SNP Database(Whitehead Institute)http:/www.broad.mit.edu

29、/snp/human/The Allele Frequency Database(the U.S.National Science Foundation)http:/alfred.med.yale.edu/alfred/index.aspJSNP(The University of Tokyo and Japan Science and Technology Agency)http:/snp.ims.u-tokyo.ac.jp/GeneSNPs(National Institute of Environmental Health Sciences)http:/www.genome.utah.e

30、du/genesnps/http:/www.ncbi.nlm.nih.gov/SNP/index.htmlSNPs in dbSNPHomoSapiensJUN 4，2003MAY 28，2004#of submissions6,818,78717,176,850#of RefSNP clusters4,145,6339,098,790(validated)512,2474,267,639#of with frequency216,920561,124#of with genotype47,728224,830http:/snp.ims.u-tokyo.ac.jp/http:/snp.cshl.org/http:/hgvbase.cgb.ki.se/