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 primer5’-labeled primer

ATP, TTP, CTP, GTPATP, TTP, CTP, GTP

Terminated fragmentsTerminated fragments

Followed by autoradiogramFollowed 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 electrophoresisNotice 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