©1999 timothy g. standish dna sequencing timothy g. standish, ph. d
TRANSCRIPT
©1999 Timothy G. Standish
DNA SequencingDNA Sequencing
Timothy G. Standish, Ph. D.
©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
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
DNA SequencingDNA Sequencing
Plasmid (or phage) with cloned DNA
fragment
Primer Binding sites
Cloned fragment
Primer
©1999 Timothy G. Standish
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….
©1999 Timothy G. Standish
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
©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 SequencingDNA SequencingWhat A SequencingWhat A Sequencing
Autorad ActuallyAutorad ActuallyLooks LikeLooks Like
A C G T
5’CTAGAGGATCCCCGGGTACCGAGCT...3’
©1999 Timothy G. Standish