Primer Template of 3 unknown sequence Dye-labeled segments of DNA. → copied from template with Denature unknown sequence four dNTPs, four ddNTPs Dye-labeled segments are applied to a DNA 三 eapiliary gel and migration subjected to electrophoresi&。 DNA sequencing can Laser beam Detector Laser be automated 27 CCT GT TTG AT GGTG GT T CCGA AAT C GG Computer-generated result after bands migrate past detector
DNA sequencing can be automated
30 40 50 60 70 80 90
Maxam-Gilbert Sequencing The Maxam-Gilbert sequencing method follows a very different strategy. Instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators,this method starts with full length,end-labeled DNA and cleaves it with base-specific reagents
Maxam-Gilbert Sequencing The Maxam-Gilbert sequencing method follows a very different strategy. Instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators, this method starts with full length, end-labeled DNA and cleaves it with base-specific reagents
(a) DNA labeled at one end with 32p 的的 Base modification Release or displacement of reacted bases Strand scission (b) 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC G GG G G G 32p 32pGpCpTp 32pGpCpTpGpCpTpAp 32pGpCpTpGpCpTpApGp 32pGpCpTpGpCpTpApGpGpTP 32pGpCpTpGpCpTpApGpGpTpGpCPCP 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpAP 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC
TO We use dimethyl sulfate (DMS)to methylate guanines.we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. Next,we use a reagent(piperidine,哌啶) that does two things:It causes loss of the mcthylated base,then it breaks the DNA backbone at the site of the lost base (the apurinic site)
TO G We use dimethyl sulfate (DMS) to methylate guanines. we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. Next, we use a reagent (piperidine，哌啶) that does two things: It causes loss of the mcthylated base, then it breaks the DNA backbone at the site of the lost base (the apurinic site)