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Dideoxynucleotides（双脱氧核苷酸） ddNTPs 是反应终止剂 可以当作正常碱基参与复制， 一旦链入DNA中，其后就不能再继续连接。 反应体系中dNTPs的浓度远高于ddNTPs(一般1：3~4)。 鸟枪测序法(shotgun sequencing)  大分子DNA被随机地“敲碎”成许多小片段，收集这些随机小片段并将它们全部连接到合适的测序载体（如M13噬菌体）；小片段测序完成后，根据重叠区计算机将小片段整合出大分子DNA序列。这就是所谓的鸟枪测序法。   What is a DNA Microarray? A DNA microarray is collection of DNA probes attached to a solid support in an orderly manner. Is a dense grid of DNA elements (often called features or cells ) What are DNA Microarrays Used For? Expression Analysis SNP genotyping Chromatin profiling Molecular bar-coding Identifying insertions DNA methylation analysis Food Safety Why use DNA Microarrays for Expression Analysis? Conventional expression analysis only allows the study of the expression of a single gene in a single experiment The highly parallel nature of microarrays allows the simultaneous study of the expression of thousands or even tens of thousands of different genes in a single experiment Microarrays allow researchers to undertake global expression analysis that is not feasible with conventional techniques Conventional Expression Analysis Expression Analysis Using Spotted DNA Microarrays Types of DNA Microarrays Spotted Arrays 点样法 --cDNA or genomic DNA clones Affymetrix arrays (genechip)原位合成法 --Oligonucleotides Homemade Tailored Cheaper????? Maximum 5000 features per cm2 Prone to variability Commercially available “Off the rack” More expensive????? Maximum 1000,000 features per cm2 Less variability Reading an array Laser scans array and produces images One laser for each color, e.g. one for green, one for red Image analysis, main tasks: Noise suppression Spot localization and detection, including the extraction of the background intensity, the spot position, and the spot boundary and size Data quantification and quality assessment Image Analysis is a book on its own: Kamberova, G. Shah, S. “DNA Array Image Analysis Nuts Bolts“. DNA Press LLC, 2002 Reading an array (cont.) Real DNA Microarray Y-fold Biol