DNA Replication
• Semiconservative DNA
replication
• Each strand of DNA acts
as a template for
synthesis of a new
strand
• Daughter DNA contains
one parental and one
newly synthesized
strand
• Bidirectional DNA
replication in E. coli
• New strands of DNA
are synthesized at the
two replication forks
where replisomes are
located
DNA Polymerase
• E. coli contains three DNA polymerases
• DNA polymerase I - repairs DNA and
participates in DNA synthesis of one strand
• DNA polymerase II - role in DNA repair
• DNA polymerase III - the major DNA replication
enzyme, responsible for chain elongation
• Synthesis of the new strand is always 5’
3’
Diagram of the replication fork
Lagging-Strand Synthesis
is Discontinuous
• Leading strand is synthesized as one
continuous polynucleotide (beginning at origin
and ending at the termination site)
• Lagging strand is synthesized discontinuously in
short pieces (Okazaki fragments)
• Pieces of the lagging strand are then joined by a
separate reaction
Diagram of lagging-strand
synthesis
• DNA pol III requires short stretch of RNA
as a Primer before it can add new
nucleotides
• DNA pol I removes these from the lagging
strand
• DNA ligase joins the Okazaki fragments
Sequencing DNA Using
Dideoxynucleotides
• Sanger method uses 2’,3’-dideoxynucleoside
triphosphates (ddNTPs) which are incorporated at
the 3’ end of a growing chain in place of a dNTP
• Since ddNTPs lack a 3’-hydroxyl group,
subsequent nucleotide addition cannot take place
• Small amounts of ddNTP’s terminate replication of
some chains at each step, leaving a set of
fragments of different lengths
The Polymerase Chain Reaction
Amplifies Selected DNA
Sequences
• The polymerase chain reaction (PCR) is used
for amplifying a small amount of DNA
• Also can increase the proportion of a particular
DNA sequence in a mixed DNA population
• PCR technique is illustrated on the next 3 slides
three cycles of the PCR reaction)
PCR needs
• Template DNA – DNA to be copied
• Taq polymerase – a heat stable DNA
polymerase
• Nucleotides – the monomers needed to
make the DNA polymer
• RNA primer – needed for polymerase to
start making DNA
• Buffer
• Thermocyler – alternates temperature
PCR Procedure
•
•
•
•
Mix all together
Heat to denature DNA – separate strands
Cool to allow RNA primers to attach
Adjust temperature to optimum for
replication
• Repeat – increase number of DNA
molecules by a factor of 2 for each cycle
DNA Amplification with the
Polymerase Chain Reaction (PCR)
5’
3’
5’
3’
3’
3’
5’
5’
Starting DNA
Template
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add
primers
(extend
primers)
5’
(anneal)
3’
3’
5’
Reverse primer
PCR Copies DNA Exponentially
through Multiple Thermal Cycles
Original DNA target region
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created