DNA Sequencing Sanger Di-deoxy method of Sequencing Manual versus Automatic Sequencing Sequencing of DNA Requires DNA amplification Labeling of DNA fragments Termination of extension Separation of fragments Detection of fragments ddNTP (one) ddNTP (one) 5-labeled primer 5-labeled primer ATP, TTP, CTP, GTP ATP, TTP, CTP, GTP Terminated fragments Terminated fragments Followed by autoradiogram Followed by autoradiogram The Reaction Mixture Sanger Method dideoxynucleotides: lack a 3 OH But have the 2 and 3 have hydrogen NO DNA EXTENSION Dideoxy key point! DNA PCR amplification is performed prior to gel electrophoresis Notice the reaction of this one reaction mixture! Separation followed by Detection Can you sequence without using radioactivity? Example of Label the ddNTPs ddGTP ddATP ddTTP ddCTP Orange Black Red Blue Automated Sequencing Efficient Does not require radioactivity The End Onto DNA Mutations