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Pathogens & PCR When a cell divides the extra DNA comes from replication
Technology
DNA replication only occurs at a specific step in the cell cycle
DNA replication is semi-
semi-conservative, one strand serves as the
template for the new strand
One of the parent strands of DNA is 3' -> 5' and the other is
5' -> 3'. To solve this replication is in opposite directions.
B.K.Kolita Kamal Jinadasa,
Jinadasa, Heading towards the replication fork, the leading strand in
Post Harvest Technology Division, synthesized in a continuous fashion, only requiring one primer.
NARA, On the other hand, the lagging strand, heading away from the
Colombo-
Colombo-15, replication fork, is synthesized in a series of short fragments
Sri Lanka. known as Okazaki fragments, consequently requiring many
primers
DNA REPLICATION
The most important enzyme of the DNA replication is DNA Polymerase I (Pol
I). Three activities are associated with DNA polymerase I;
DNA strands
5' to 3' elongation (polymerase activity)
3' to 5' exonuclease (proof-
(proof-reading activity)
5' to 3' exonuclease (repair activity)
For example,
example, if we amplify a genetical marker of E.coli,
E.coli, we are sure that only
E.coli will be amplify and the others bascterias don’
don’t amplify.
amplify.
1. Divalent cation,
cation, magnesium or manganese ions;
ions; generally Mg2+ is used,
used, but
Mn2+ can be utilized for PCR- PCR-mediated DNA mutagenesis,
mutagenesis, as higher Mn2+
Thermocycler concentration increases the error rate during DNA synthesis.
synthesis.
1
PCR STEPS PCR steps
The PCR usually consists in a series of 20 to 35 cycles.
cycles. PCR is carryed out
in three steps:
steps:
PCR
One PCR cycle consists of the following steps General PCR
Initialization.
Initialization. The mixture is heated at 96°
96°C for 5 minutes to ensure that
the DNA strands as well as the primers have melted.
melted.
Melting,
Melting, where it is heated at 96°
96°C for 30 seconds.
seconds. For each cycle,
cycle, this is
usually enough time for the DNA to denature.
denature.
Exponencial amplification
TYPES OF PCR
There are a lot of types of PCR :
1. Nested PCR
2. Inverse PCR
3. RT-
RT-PCR (Reverse Transcription PCR)
4. Quantitative real-
real-time PCR
5. Multiplex-
Multiplex-PCR
6. Asymmetric PCR
7. "Hot-
"Hot-start" PCR
8. PCR-
PCR-RFLP
Because both strands are copied during PCR, there is an 9. PCR-
PCR-ELISA
exponential increase of the number of copies of the gene.
Supposing that there is only one copy of the wanted gene before the
cycling starts,
starts, after one cycle,
cycle, there will be 2 copies, after two
cycles, there will be 4 copies, three cycles will result in 8 copies and
cycles,
so on.
on.
2
APLICATIONS OF PCR
PCR is commonly used in medical and biological research labs for a variety of
tasks,
tasks, such as the detection of hereditary diseases,
diseases, the identification of
species,
species, diagnosis of infectious diseases,
diseases, cloning of genes,
genes, paternity testing,
testing,
DNA computing and detection of pathogens in food.food.
Some of foodborne pathogens that we can find in food are: Escherichia coli, coli,
Salmonella spp,
spp, Shigella spp,, Staphylococcus aureus,, Vibrio choleraea and Vibrio
parahaemolyticus.