DNA REPLICATION

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)

„ The replication of DNA in vitro is named polymerase chain reaction

reaction (PCR). PCR
is used to amplify specific regions of a DNA strand.
strand. With this tecnique we can
have a lot of copy of a determinate region.
region.

„ PCR is especific bacause we amplify specific regions of bacterias, animals,

animals,
plants,
plants, viruses...
viruses... Thes regions are named molecular markers.
markers.

„ 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.

„ PCR was invented by Kary Mullis.

Mullis. At the time he thought up PCR in 1983,
Mullis was working in Emeryville,
Emeryville, California for Cetus Corporation .

„ The polymerase chain reaction was introduced to the scientific community at a

conference in October 1985

Kary Mullis PCR: POLYMERASE CHAIN REACTION

.

„ PCR, as currently practiced,

practiced, requires several basic components:
components:
Nobel Prize for chemistry in 1993 1. DNA template that contains the region of the DNA fragment to be amplified.
amplified.

2. Taq polymerase (or another DNA polymerase with a temperature optimum at

around 70°
70°C), a DNA polymerase,
polymerase, used to synthesize a DNA copy of the region to
be amplified.
amplified.

1. Deoxynucleotide triphosphates, (dNTPs)

dNTPs) from which the DNA polymerase
builds the new DNA.

1. Buffer solution, which provides a suitable chemical environment for optimum

activity and stability of the DNA polymerase.
polymerase.

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:

1. Denaturation at 94°94°C : During the denaturation,

denaturation, the double strand melts
open to single stranded DNA, all enzymatic reactions stop (for
(for example:
example: the
extension from a previous cycle).
cycle).

2. The denaturation is followed by the annealing step.

step. In this step the reaction
temperature is lowered so that the primers can attach to the single-
single-stranded
DNA template.
template. The temperature at this step depends on the Tm of the
primers (see above),
above), and is usually between 50-
50-64°
64°C for 20-
20-40 seconds.
seconds.

3. The annealing step is followed by an extension/

extension/elongation step during
which the DNA polymerase copies the DNA template,
template, starting at the
primers annealed to both of its strands.
strands. The temperature at this step
depends on the DNA polymerase used.
used. Taq polymerase has a temperature
optimum of 70- 74°C; thus,
70-74° thus, in most cases, during the extension a
temperature of 72°
72°C is used

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.

„ Annealing by heating at 68°

68°C for 30 seconds.
seconds. Stable bonds are only formed
when the primer sequence exactly fits the template sequence,
sequence, and on that
short piece of double-
double-stranded DNA (template
(template and primer), the
polymerase can attach and start copying the template.
template. Once this extension
has created a longer double-
double-stranded DNA segment,
segment, the Tm of this
double-
double-stranded region is now greater than the annealing or extension
temperature.

„ Elongation by heating 72°

72°C for 45 seconds:
seconds: This is the ideal working
temperature for the polymerase.
polymerase. The combined hydrogen bonds between
the extended primer and the DNA template are now strong enough to
withstand forces breaking these attractions at the higher temperature.
temperature.
Primers that are on positions with no exact match, melt away from the
template (because of the higher temperature)
temperature) and are not extended.

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.

„ The detection of foodborne pathogens is very important bacause this

pathogens can cause very important illnes.
illnes.

„ 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.