分子生物学（杨君）lecture11 Methods.pptVIP

9. Sequencing Two ways for sequencing: DNA molecules (radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. (Maxam and Gilbert chemical method, not widely used) Chain-termination method (Sanger’s method, widely used) Walter Gilbert Nobel Prize 1980 RNA word Exon intron Frederick Sanger Nobel Prize 1958 Nobel Prize 1980 Paul Berg Nobel Prize 1980 Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 Chain-termination method (链终止法) ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 Chain-termination method (链终止法) The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule. Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 Chain-termination method (链终止法) If one ddGTP is added to 100 dGTP, DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP Tell from the gel the position of each G Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 DNA sequencing gel Four separate reactions dNTP+ ddGTP dNTP+ ddATP dNTP+ ddCTP dNTP+ ddTTP Each ddNTP carries a fluorescence group, allowing us to “Read” the sequence directly from the gel. Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 Automatic sequencer Fluorescence labeled ddNTP Polymerase catalyzed Ch20技术 相互作用 蛋白技术 核酸技术 结构分析 小 结 练 习 Shotgun sequencing of a bacterial genome (鸟枪法测细菌的基因组) The bacterial genome was randomly sheared into many random fragments with an average size of 1 kb, and cloned intro a vector.[Prepare what you are going to shot] Individual recombinant DNA clones are randomly picked to prepare DNA for sequencing on an automated sequencer. [shot] In use of shotgun sequencing strategy, multiple sequence coverage is required to obtain all genome sequence automated shotgun sequencing (obtain sequence) then the subsequent use of computer to assemble the different sequences Ch