单核苷酸多态性snp汕头大学医学院医学细胞生物学精品课程课件

Gene Variations single nucleotides polymorphism & copy number variation 刘戈飞 汕头大学医学院遗传学与细胞生物学教研室 075488900497Genetic Variations Chromosome numbers Segmental duplications, Copy number variation Translocations Inversion Sequence Repeats Transposable Elements Short deletions and insertions Tandem Repeats Nucleotide Insertions and Deletions (Indels) Single Nucleotide Polymorphisms (SNPs) Mutations Sizable Minor Structural Sequence Genetic Markers Morphological markers Cytological markers Biochemical and physiological markers Molecular markers 1980, RFLPs (restriction fragment length polymorphisms) 1985, STRs (short tandem repeats, mini-satellites) 1990s, SNPs (single nucleotide polymorphisms) 2000s, CNV (copy number variation) A C G T G T C G G T C T T A A A Maternal chromosome A C G T G T C C G T C T T A A A Paternal chromosome A 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 chromosome A C G T G T C C G T C T T A A A Maternal chromosome A C G T G T C C T A C T T A A A Paternal chromosome The position of the SNP is indicated by the box. Individual 1 is heterozygous, while individuals 2 and 3 are homozygous. Individual 1 Individual 2 Individual 3 SNP Single nucleotide polymorphism (SNP) 在基因组中，不同个体的DNA序列上的单个碱基的差异被称作单核苷酸多态性。 1/1000 Estimated between any 2 individuals (3 m) 10 m in the whole populations Single nucleotide polymorphism (SNP) SNP Effects SNPs in genes In coding regions (possible protein structure changes) Synonymous substitutions (同义) Missense substitutions (错义) Nonsense substitutions (终止) In coding and non-coding regions Change of gene expression (by diverse binding various factors) Yield Timing Alternative splicing SNPs in regulatory regions Change of gene expression SNPs in non-regulatory intergenic regions Can be used as genetic markers 1234 121243 HapMap 国际人类基因组单体型图计划 Towards genome variations 人类的所有群体中大约存在一千万个SNP位 点，其中稀有的SNP位点的频率至少有1% 。 相邻SNPs的等位位点倾向于以一个整体遗 传给后代。位于染色体上某一区域的一组 相关联的SNP等位位点被称作单体型 (haplotype)。 大多数染色体区域只有少数几个常见的单 体型(每个具有至少5%的频率)，它们代表 了一个群体中人与人之间的大部分多态性 。一个染色体区域可以有很多SNP位点， 但是只用少数几个标签SNPs，就能够提供 该区域内大多数的遗传多态模式。 HapMap的构建分为三个步骤：a在多个个体的DNA样品中鉴定 单核苷酸多态性SNPs；b将群体中频率大于1%的那些共同遗传 的相邻SNPs组合成单体型；c在单体型中找出用于识别这些单 体型的标签SNPs。通过对图中的三个标签SNPs进行基因分型， 研究者可以确定每个个体拥有图示的四个单体型中的哪一个。 We are so young! with limited number of ancestors with a few (thousands) of generations with only a few recombination events 我们非常年轻 人类进化史上曾有一大瓶颈（约6-15万年前） 通过“瓶颈”的人类祖先群体很小（仅有万余人） 现代人类仅经过少数几千个时代（约30005600代） “遗传重组”数目有限 Human genome is composed of “blocks” 单体型的起源 Methods and technologies in SNP studies Discovery (Find SNPs) Validation (A common one or rare one) Genotyping (Frequency in population) Consideration: Call rates Flexibility Throughput Cost Fundamental approaches large-scale sequencing based: genomic-alignment(GA), reduced representation shotgun(RRS) PCR based: common PCR hybridization based: DNA chips Genomic DNAmRNA BAC librarycDNA library RRS (reduced representation shot-gun) library or sampling BAC overlapShotgun overlapEST overlap Sequence overlap SNP discovery GTTTAAATAATACTGATCA GTTTAAATAATACTGATCA GTTTAAATAGTACTGATCA GTTTAAATAGTACTGATCA How to discover SNPs Base-calling Quality determination Contig assembly Sequence viewing Polymorphism tagging Polymorphism reporting Individual genotyping Polymorphism detection PolyPhred Consed Analysis Sequence PhredPhrap Amplify DNA 53 Discovering SNPs by Sequencing Phylogenetic analysis ATAGACG ATACACG ATAGACG ATACACG ATAGACG ATACACG HomozygotesHeterozygote SNP检定 Genotyping 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)、Pyrosequencing Throughput SNP screening of certain genes 5UTR exons 3UTR-1000-1 regulation region Genes, Samples, Phenotypes Primers design and PCR Directly DNA sequencing Statistical Analysis SNP raise the resolution of genetic analysis Pharmacogenomics Personalize medicine 2|JANUARY 2007|VOLUME 8 /reviews/genetics Science,2004 23 JULY 2004,305:525 Forty-three authors used the DNA from 270 individuals from the 4 HapMap populations. Overall, the authors found 1,447 discrete, heterogeneously distributed, copy number variable regions (CNVRs), which cover 12% of the human genome. They found that 24% of CNVRs are associated with segmental duplications. CNVRs contain different classes of functional elements. many CNVs preferentially lie outside genes. genes that are involved in cell-adhesion functions, sensory perception of smell and response to chemical stimuli are enriched within CNVs. Conversely, cell signalling and proliferation, as well as kinase-and phosphorylationrelated categories were underrepresented among CNVs. Interestingly, ultraconserved elements are strongly excluded from these regions. CNV has effects on SNP genotype patterns. SNP has the ability to identify linked CNV. Both types of variation will need to be collected and analysed systematically if we are to understand the genetic basis of human disease. The authors call for standard assessment of CNV in all studies of the genetic basis of phenotypic variation, and for an international effort to continue to characterize and catalogue structural genomic variation. 26,628 clones534500 SNPs phenotype: modify drug response predispose to or cause disease polymorphism: population genetics genome wide gene regulation variation Effects of CNV Genome-wide array-based array- CGH: Clone-based(1Mb), oligonucleotide- based(30kb) SNP array (signal intensity, genotyping) sequence-assembly comparison Targeted PCR-based MAPH, MLPA, QMPSF: mutiplex, up to 40 regions per time real-time qPCR Hybridization-based FISH, Southern blotting Computation approaches Methods to identify CNV Methods to identify CNV: array-CGH array-based CGH Methods to identify CNV: array-CGH representational oligonucleotide microarray analysis, ROMA multiplex amplifiable probe hybridization, MAPH Methods to identify CNV: targeted PCR-based Multiplex ligation-dependent probe amplification, MLPA Methods to identify CNV: targeted PCR-based Quantitative multiplex PCR of sh