Download Pathogenic bacteria Genomic DNA extracted from

BIOL 4102 – Week 1 (Polymerase Chain Reaction)
Week 1: Molecular Cloning of the SSB gene into the pCDF plasmid for
protein expression.
Introduction – SSB gene from a specific species of pathogenic bacterium has
already been cloned into the pUC57 plasmid.
SSB gene
Pathogenic bacteria
PCR using primers
specific for the SSB
gene was carried out
to amplify SSB DNA
Genomic DNA
extracted from
pathogenic bacteria
SSB gene was inserted into
the pUC57 plasmid by
molecular cloning
Ampilillin resistance
gene
pUC plasmid
Ori
SSB gene
Week 1 – Learning the concept of PCR by amplifying E.coli SSB DNA
from pUC57 plasmid.
Overview:
• To learn the concept and technique of PCR, SSB gene cloned into the pUC57
plasmid will be amplified using forward and reverse primers (shown in red).
•
The amplified SSB PCR product will be analyzed using agarose gel
electrophoresis.
Ampicillin resistance gene
Ori
pUC57 plasmid
T7
SSB gene
promoter
T7
terminator
PCR followed by agarose gel
electrophoresis to analyze the
DNA amplified by PCR.
DNA size
marker
PCR product
Gel Lanes
5 kb
2 kb
1 kb
0.5 kb
SSB
0.2 kb
Sofia Origanti and Edwin Antony, 2015
(Expected DNA size of
SSB – 0.5kb)
Week 1 – Learning the concept of PCR by amplifying DNA inserted into
the pCDF plasmid.
Overview (cont’d.) : PCR- Schematic
Plasmid DNA +
PCR reaction
tube
PCR machine
PCR steps
Cycle 1
Forward & Reverse +
T7 primers
Nucleotides +
DNA polymerase
5’
3’
3’ SSB gene section of the plasmid
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
Denaturation at 98°C separation of the two strands
of double stranded DNA
Annealing at 55°C –pairing of the
forward and reverse primers (in
red) with the complementary
sequence on SSB DNA
Elongation at 72°C –DNA
polymerase (in blue) moves in the
3’ to 5’ direction of the template
strand and adds nucleotides to the
primers (in pink) in a complementary fashion
2 copies of SSB DNA at
the end of first cycle
Cycle 2
Exponential doubling with each
cycle- 2 copies of SSB DNA
become 4 at the end of second
cycle. 4 copies become 8 with
cycle 3 and so on.
Sofia Origanti and Edwin Antony, 2015