DNA Sequencing Scenario
DNA replication and chain termination
P
P
P
P
H
P
5
P
P
P
P
P dATP
ddTTP
ÖH OH
No OH group = chain
termination at T residue
T
Primer strand
T
C
A
G
A
A
G
T
C
T
A
The 3 hydroxyl (OH) group
interacts with the phosphate
group on the incoming
C nuclueotide to form a
phosphodiester bond
T
Template strand
3
P
P
P
P
P
P
P
P
Adapted from Lehninger Principles of Biochemistry 4th Edition
Nucleosides & Nucleotides
Taken from: http://en.wikipedia.org/wiki/Nucleotide
Nucleoside = a heterocyclic base (A, G, C, U or T) and a five carbon sugar
(pentose) either ribose for RNA or deoxyribose for DNA
Nucleotide = a heterocyclic base and a pentose sugar with one or more phosphate
groups (mono-, di- or tri-phosphate).
Nucleotides are the monomers of nucleic acids, with three or more bonding
together in order to form a nucleic acid.
Nucleotide structure continued
O-O
O-
O-
P O P O P O CH2
O
O
O
H H
O-O
O-
O-
O
O
H H
OH
P O P O P O CH2
O
O
-O
O
With a ribose sugar (OH at C2 &
C3) used to synthesise RNA.
Deoxynucleoside tri-phosphate (dNTP)
With a deoxyribose sugar (OH at C3)
used to synthesise DNA.
H
O-
P O P O P O CH2
O
Base
H H
OH
O-
Nucleoside tri-phosphate (NTP)
OH
H H
O-
Base
O
O
H H
H
O
Base
H H
H
Dideoxynucleoside tri-phosphate (ddNTP)
With a dideoxyribose sugar (no OH
groups) used for the Sanger method
(dideoxy) sequencing.
Nucleotides are joined together by phosphodiester bonds
phosphodiester bond
http://en.wikipedia.org/wiki/Phosphodiester_bond
Phosphodiester bonds require a hydroxyl group (OH) to be present on the 3´
carbon to react with a phosphate on in 5 carbon of the sugar residue of the next
nucleotide.
If no 3´ hydroxyl group is available no further nucleotides can join the DNA strand
and the chain terminates. This is how dideoxynucleotides prevent chain elongation
– they lack a 3´ OH group on the sugar.
Outline of Fluorescent Dideoxy DNA Sequencing
Components of the reaction mix
• DNA template
• DNA primer
• DNA polymerase -Taq polymerase
• Mixture of deoxy (dNTPs) & fluorescently labelled
dideoxy nucleotides (ddNTPs)
20 – 35 cycles
Apply to a capillary gel &
subject to electrophoresis
Computer
analysis –
base calling
The fragments are detected after excitation
Fluorescent peaks and bases
of the labelled ddNTPs by a laser
are visualised and analysed
The sequencing reaction continued
Components in the reaction mix
Taq polymerase
5
PRIMER
3
TEMPLATE
OH
AGTCTATATC
+ dATP, dCTP, dGTP, dTTP,
+ ddATP, ddCTP, ddGTP, ddTTP
Termination products obtained
Remember that the sequence obtained with be complementary to the template
sequence!
PRIMER
T
PRIMER
TCAGAT
PRIMER
TC
PRIMER
TCAGATA
PRIMER
TCA
PRIMER
TCAGATAG
PRIMER
TCAG
Therefore our new 5
3 complementary sequence to our original template
sequence reads TCAGATAG.