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DNA SequencingDNA Sequencing
Timothy G. Standish, Ph. D.
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©1999 Timothy G. Standish
H
P
O
OH
OH
HO
O
O
CH2
NH2
N
N N
N
Sugar
Base
Phosphate
3’
5’
2’
1’4’
DideoxynucleotidesDideoxynucleotides 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
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SU
GA
R-P
HO
SP
HA
TE
BA
CK
BO
NE
H
P
O
HO
O
O
CH2
HOH
P
O
O
HO
O
O
CH2
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
B A
S E
S
2’3’2’3’dideoxy-dideoxy-
nucleotidesnucleotidesTerminateTerminate
DNADNAReplicationReplication OH
P
O
HO
O
O
CH2
HO
O
H 2N
NHN N
N H
H OH
P
O
OH
O
O
CH2
CH 3
O
O
HNN
OH
H
P HO
O
O
CH2
HO
N
O
H 2N
N
H2O
2’3’did
eoxynu
cleotide
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DNA SequencingDNA 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
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DNA SequencingDNA Sequencing
Plasmid (or phage) with cloned DNA
fragment
Primer Binding sites
Cloned fragment
Primer
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The ddATP ReactionThe ddATP Reaction
5’TTATCG3’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 comeon!
Pol.5’TTATCGTACCATGACTAGA
NotAgain!
Pol.5’TTATCGTACCATGACTAGATGCGATA
Agggg….
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DNA SequencingDNA 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
ddTTPddCTP ddGTPddATP
Rea
d 5’
to 3
’ fr
om b
otto
m to
top
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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 SequencingDNA SequencingWhat A SequencingWhat A Sequencing
Autorad ActuallyAutorad ActuallyLooks LikeLooks Like
A C G T
5’CTAGAGGATCCCCGGGTACCGAGCT...3’
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