1. DNA Sequencing Scenario

2. DNA replication and chain termination
Adapted from Lehninger Principles of Biochemistry 4th Edition 5´ P T A C G 3´ ÖH OH
dATP
ddTTP
No OH group = chain termination at T residue P H T
Primer strand
The 3´ hydroxyl (OH) group interacts with the phosphate group on the incoming nuclueotide to form a phosphodiester bond
Template strand

3. Nucleosides & Nucleotides
Taken from:
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.

4. Nucleotide structure continued
Base H OH
CH2 -O P O-
Nucleoside tri-phosphate (NTP)
With a ribose sugar (OH at C2 & C3) used to synthesise RNA. O
Base H OH
CH2 -O P O-
Deoxynucleoside tri-phosphate (dNTP)
With a deoxyribose sugar (OH at C3) used to synthesise DNA. O
Base H
CH2 -O P O-
Dideoxynucleoside tri-phosphate (ddNTP)
With a dideoxyribose sugar (no OH groups) used for the Sanger method (dideoxy) sequencing.

5. Nucleotides are joined together by phosphodiester bonds
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.

6. 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 of the labelled ddNTPs by a laser
Fluorescent peaks and bases are visualised and analysed

7. The sequencing reaction continued
Components in the reaction mix
Taq polymerase 5´
PRIMER OH
+ dATP, dCTP, dGTP, dTTP,
+ ddATP, ddCTP, ddGTP, ddTTP 3´
TEMPLATE
AGTCTATATC
Termination products obtained
Remember that the sequence obtained with be complementary to the template sequence!
PRIMER T
PRIMER
TCAGAT
PRIMER
TCAGATA
PRIMER TC
PRIMER
TCAGATAG
PRIMER
TCA
TCAG
PRIMER
PRIMER
TCAGA
Therefore our new 5´  3´ complementary sequence to our original template sequence reads TCAGATAG.