©1999 Timothy G. Standish DNA Sequencing Timothy G. Standish, Ph. D.

©1999 Timothy G. Standish H P O OH HO O O CH 2 NH 2 N N N N Sugar Base Phosphate 3’ 5’ 2’ 1’4’Dideoxynucleotides DNA Sequencing using the Sanger method involves the use of 2’3’-dideoxynucleotide triphosphates in addition to regular 2’-deoxynucleotide triphosphates Because 2’3’-dideoxynucleotide triphosphates lack a 3’ hydroxyl group, and DNA polymerization occurs only in the 3’ direction, once 2’3’-dideoxynucleotide triphosphates are incorporated, primer extension stops H 2’3’-dideoxynucleotide monophosphate 2’-dideoxynucleotide monophosphate

SUGAR-PHOSPHATE BACKBONE H P O HO O O CH 2 HOH P O O HO O O CH 2 H P O OH HO O O CH 2 NH 2 N N N N O O N NH N N N O NH 2 N B A S E S 2’3’ dideoxy- nucleotides Terminate DNA Replication O H P O HO O O CH 2 HO O H2NH2N N HN N N H HOH P O O O CH 2 CH 3 O O HN N OH H P HO O O CH 2 H O N O H2NH2N N H2OH2O 2’3’dideoxynucleotide

©1999 Timothy G. Standish DNA Sequencing In DNA sequencing reactions all the basic components needed to replicate DNA are used 4 reactions are set up, each containing: –DNA Polymerase –Primer –Template to be sequenced –dNTPs –A small amount of one ddNTP ddATP, ddCTP, ddGTP, ddTTP As incorporation of ddNTPs terminates DNA replication, a series of fragments is produced all terminating with the ddNTP that was added to each reaction

©1999 Timothy G. Standish DNA Sequencing Plasmid (or phage) with cloned DNA fragment Primer Binding sites Cloned fragment Primer

©1999 Timothy G. Standish The ddATP Reaction 5’TTATCG 3’AATAGCATGGTACTGATCTTACGCTAT5’ 5’TTATCGTACCATGACTAGATGCGA 5’TTATCGTACCA 5’TTATCGTACCATGACTA 5’TTATCGTA 5’TTATCGTACCATGA 5’TTATCGTACCATGACTAGATGCGATA 5’TTATCGTACCATGACTAGA Pol. 5’TTATCGTA Let me Through! Pol. 5’TTATCGTACCATGA Oh come on! Pol. 5’TTATCGTACCATGACTAGA Not Again! Pol. 5’TTATCGTACCATGACTAGATGCGATA Agggg….

©1999 Timothy G. Standish DNA Sequencing Products from 4 reactions each containing a small amount of a dideoxynucleotide are loaded onto a gel Polyacrlyamide gels capable of separating fragments differing in size by only one base High concentrations of urea are used to prevent formation of double-stranded DNA or secondary structures Because polymerization goes 5’ to 3’ shortest fragments are 5’ compared to longer fragments which are in the 3’ direction ddTTPddCTPddGTPddATP Read 5’ to 3’ from bottom to top

©1999 Timothy G. Standish To read the autorad it is important to start at the bottom and work up so that it is read in the 5’ to 3’ direction DNA Sequencing What A Sequencing Autorad Actually Looks Like A C G T 5’ CTAGAGGATCCCCGGGTACCGAGCT... 3’

©1999 Timothy G. Standish