DNA Sequencing Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

What to Sequence? The RT-PCR product has been inserted into the pCR4-TOPO vector Our goal -Sequence the insert DNA

We Supplied Template DNA: Your plasmid Primer DNA: T3 or T7

Frederick Sanger Discovered DNA sequencing by chain termination method Nobel Prize 1 (1958) –Complete amino acid sequence of insulin Nobel Prize 2 (1980) –For DNA sequencing

DNA Polymerase Action ATTAACCC TCACTAAAGG T3 Primer GACTAGTCCT GCAGGTTTAA AGGAATTCGC CCTT DNA Polymerase DNA Sequencing exploits the DNA polymerase activity for deciphering DNA sequence Modern DNA sequence use PCR technology in sequencing

One primer PCR Reaction Template DNA Primer DNA Polymerase dATP dGTP dCTP dGTP Nucleotides NEW STRAND

Dideoxy Nucleotides Lack an -OH group at the 3-carbon position Cannot add another nucleoside at that position Prevent further DNA synthesis

Dideoxy nucleotides Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension Are added in small proportion –dATPddATP –dGTPddGTP –dCTPddCTP –dTTPddTTP

Chain Termination

All Possible Terminations

Polyacrylamide Gel Electrophoresis Separates fragments based on size

DNA Sequence Files

Good or Bad?

Do not forget the other strand T7 Primer GGG ATATCACTCA GCATAATTGTTAAGTGACC

GenBank The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.

Shotgun Sequencing Involves fragment assembly using computer algorithms Contigs