• 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

DNA Replication

• 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

Make copies (extend primers)

Starting DNA Template

5’

5’

3’

3’

5’

5’

3’

3’

Add primers (anneal) 5’3’

3’5’

Forward primer

Reverse primer

DNA Amplification with the Polymerase Chain Reaction (PCR)

Separate strands

(denature)

5’

5’3’

3’

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

PCR Copies DNA Exponentially through Multiple Thermal Cycles

Original DNA target region

Thermal cycleThermal cycleThermal cycle