ch4DNAsequencingandassembling基因组学.pptVIP

Genomics Chapter 4 DNA sequencing and assembling Contents The basic methodology for DNA sequencing Assembly of a contiguous DNA sequence Platform of genome sequencing New DNA-Sequencing Technologies Advancing 4.1 The basic methodology Chain termination DNA sequencing (Sanger et al, 1977): the sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions; Chemical degradation method (Maxam and Gilbert, 1977): the sequence of a double-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions. 4.1.1 chain termination sequencing A small amount of a dideoxynucleotide (ddNTP) DNA polymerase for chain temination sequencing (Sequenase)—T7 DNA polymerase 1) high processivity 2) negligible or zero 5‘?3’exonuclease activity 3) negligible or zero 3‘?5’ exonuclease activity Single-stranded DNA template p166 Chain termination DNA sequencing 用于测序的DNA多聚酶 Preparation of single-strand DNA template Denaturation after be cloned in plasmid M13 vector Phagemid vector PCR p168 4.1.2 Chemical degradation method (i) Label the 3ends of DNA with 32p. (ii) Separate two strands, both labelled at 3ends. (iii) Divide the mixture in four samples, each treated with a different reagent having the property of destroying either only G, or only C, or A and G or T and C ‘. The concentration of reagent is so adjusted that 50% of target base is destroyed, so that fragments of different sizes having 32p are produced. (iv) Electrophoreses each of the four samples in four different lanes of the gel. 4.1.3 Automated sequencing thermal cycle sequencing (热循环测序) Fluorescent primers are the basis of automated sequence reading Capillary Electrophoresis (毛细管电泳, CE) instead of Polyacrylamide Gel Electrophoresis (聚丙烯酰胺凝胶电泳, PAGE)： Applied Biosystems 美国应用生物系统公司（原Perkin-Elmer公司） ABI3700型毛细管电泳自动测序仪 Thermal cycle sequencing Two adv