基因工程第六章DNASequence答案.ppt

Chapter 6DNA Sequencing ;;;;Sanger dideoxy-mediated chain-termination Maxam-Gilbert chemical degradation;;Maxam-Gilbert DNA sequencing by chemical degradation 化学降解法;1. Basic principle;Reagents for MG DNA sequencing;;;;;;; AC G T+C C;2. Procedure ; AC G T+C C;3. Notes specificity length (200-250bp ) it is better to read Maxam and Gilbert’s article before action;第二节 dideoxy-mediated chain-termination method;1. Core principle;;;;;;;;;;;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;A: dNTP + ddATP;A: dNTP + ddATP;2. Related strategy ;(2) Template ;(3) Primer;E1 S K….…....S H3 ;E1 S K….…....S H3 ; MCS; MCS;3. Procedure;(1) Full manual operation ;Brief protocols;;每一组样品所给出的条带数目有限， 由此读出的核苷酸序列也有限;;;Lane A: dNTP + ddATP Lane C: dNTP + ddCTP Lane G: dNTP + ddGTP Lane T: dNTP + ddTTP 2 loading ;(2) PCR reaction;(2) PCR reaction;Brief protocols;PCR procedure;T T T A A G T G A A A A A A C A G C T* T A A A T T C C A A A A ;(3) Automatic sequencing;Core principle;3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’;PCR procedure;PCR reaction ;;for sequencing a DNA fragment ;1. Primer walking;2. Random clones ;EI S K….…....S H3 ;2. Random clones ;Sn=1-(1-?/L)n;if;if;3. Fixed cloned; 4. Deletion clones;Exonuclease III;第四节 RNA端点分析 1. S1酶作图;3’AGCUCGUAGCAUGCAU5’;T T T A A G T G A A A A A A C A G C T* T A A A T T C C A A A A ; Solexa SOLiD 100MB 1GB 4G 250bp 35bp 35bp ;2005年底，罗氏诊断公司和454公司推出了——Genome Sequencer 20 System (GS 2