第二章基因组的变化.ppt

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 1 2 3 4 1 2 1 2 4 3 早期SNP检测技术 限制性酶切技术 — RFLP 2. 识别异源双链技术 DHPLC（变性高效液相层析） DGGE（变性梯度凝胶电泳） SSCP（单链构象多态性） EMC（错配的酶促切割法） CCM（错配的化学切割法） 3. 同源杂交技术— ASO (Allele Specific Oligonucleotides) 常用SNP检测技术 基于PCR－引物延伸 Pyrosequencing、MassArray Fluorescence Polarization、SNuPe SnapShot 基于探针杂交 TaqMan、Invader、BeadArray、 Parallele、 SNPlex、Chip (Affymetrix) Allele specific prime extension DNA 提取 自动序列分析及等位基因信息的获得 Primer MassArray （1） 目标序列的扩增 第一次纯化和SNP位点延伸反应 点样 MALDI-TOF 质谱测定 Allele 1 Allele 1 Allele 2 Allele 2 +Enzyme +ddATP +dCTP/dGTP/dTTP Allele 1 Unlabeled Primer (23mer) T C T Extended Primer (24mer) T C T A T G A Allele 2 Unlabeled Primer(23mer) A C T Extended Primer (26-mer) A C T MassArray （2） TaqMan Application of DNA P Linkage analysis Indirect gene diagnosis Paternity test Analyzing the origin of extra chromosome 23 14 21 34 21 34 56 35 36 45 12 23 25 13 15 23 14 21 24 31 34 56 35 46 45 12 25 15 13 23 D2S123 D3S132 UNLINKED LINKED Linkage Analysis Indirect Gene Diagnosis Known disease genes - large genes - different disease-causing mutation Unknown disease genes Indirect Gene Diagnosis M1 M2 M1 1 12 1 1 3 1 11 2 M2 2 13 3 3 2 3 23 1 XAY XAXa XaY XaY XAY XaY ? ? XAXa XAY 2/5 Ⅰ Ⅱ 1 2 2 1 1 3 4 5 6 Ⅲ 2/4 3/5 2/3 4/5 2/5 3/4 4/5 2/5 2 2/4 3/5 3/4 2/2 2/5 5/5 Unknown disease genes (mapped) Indirect Gene Diagnosis M1 1 23 2 2 4 4 12 2 XAY XAXa XaY XaY XAY XaY ? ? Disease gene Marker 1 Recombination ? M1 1 23 12 2 XAY XAXa XaY XaY ? ? Indirect Gene Diagnosis Premise Family