1. Matthew 13:17
17 For verily I say unto you, That many prophets and righteous men have desired to see those things which ye see, and have not seen them; and to hear those things which ye hear, and have not heard them.

2. DNA Sequencing
Timothy G. Standish, Ph. D.

3. Sequenced Genomes
Over the past two years large-scale sequencing of eukaryotic genomes has become a reality
Currently the sequencing of at least 4 multicelled eukaryotic genomes has been completed:
1998 Caenorhabditis elegans - 8 x 107 bp - A nematode worm
2000 Homo sapiens - 3 x 109 bp - Humans
2000 Arabidopsis thaliana x A plant related to mustard
2000 Drosophila melanogaster x 108 bp - Fruit flies

4. New Technology
Rapid sequencing of large complex genomes has been made possible by:
Foundational work done over many years and…
Dramatic improvement in DNA sequencing technology over the past few years
In this presentation we will look at both the basic principles of DNA sequencing and how techniques have been refined to yield the dramatic results we now see

5. Basic Principles

6. 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
2’-dideoxynucleotide
monophosphate H P O OH HO
CH2
NH2 N
Sugar
Base
Phosphate 3’ 5’ 2’ 1’ 4’ H
2’3’-dideoxynucleotide
monophosphate

7. 2’3’ dideoxy-nucleotides Terminate DNA ReplicationH OH P O
CH2
CH3 HN N
SUGAR-PHOSPHATE BACKBONE H P O HO
CH2 OH
NH2 N NH
B A S E S O H P HO
CH2
H2N N HN H P HO O
CH2 N
H2N
H2O
2’3’dideoxynucleotide
2’3’ dideoxy-nucleotides Terminate DNA Replication

8. 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

9. Plasmid (or phage) with cloned DNA fragment
DNA Sequencing
Cloned fragment
Primer
Primer Binding sites
Plasmid (or phage) with cloned DNA fragment

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

11. 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
ddTTP
ddCTP
ddGTP
ddATP
Read 5’ to 3’ from bottom to top

12. DNA Sequencing What A Sequencing Autorad Actually Looks Like
A C G T
DNA Sequencing What A Sequencing Autorad Actually Looks Like
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
5’CTAGAGGATCCCCGGGTACCGAGCT...3’

13. The
End