1. POLYMERASE CHAIN REACTION TECHNIQUES
(1) Standard PCR:
@ Kary Mullis invented the PCR in 1983 (USA)
and synthesized short chains of DNA. Given
Nobel Prize in Chemistry in 1993.
@ He invented a method of amplifying DNA
through repeated cycles of duplication driven
by DNA polymerase enzyme.
@ PCR technique used an automated process
for DNA amplification & separation of the
DNA strands.

3. PCR Components and Reagents:
@ DNA template that contains the DNA
fragment to be amplified.
@ Two Primers that are complementary to the
3' (three prime) ends of each of the sense
and anti-sense strand of the DNA target.
@ Deoxynucleoside triphosphates (dNTPs), the
building-blocks from which DNA polymerase
synthesizes a new DNA strand.

5. @ Taq DNA polymerase with a temperature
optimum at around 70 °C.
@ Buffer solution, providing a suitable
chemical environment for optimum activity
and stability of the DNA polymerase.
@ Divalent cations (magnesium or manganese)
for DNA synthesis
@ Monovalent cation (potassium) for DNA
synthesis

7. PCR Principle:
@ PCR is a biochemical technology to amplify
a single or a few copies of a piece of DNA
to generate millions of copies of a certain
DNA sequence.
@ The technique depends on thermal cycling,
with repeated heating and cooling to melt
and amplify (replicate) the DNA.
@ Repeated amplification of DNA requires
short DNA fragments (Primers) and a heat-
stable DNA polymerase (Taq polymerase)
obtained from bacterium Thermus aquaticus.

8. A thermal cycler for PCR

9. @ This DNA Taq polymerase enzyme assembles
a new DNA strand from DNA nucleotides
primers by using the single stranded
DNA present in the reaction as a template.
@ Thus DNA synthesis is initiated and a chain
of reactions develops, resulting in massive
DNA amplification.

11. PCR Procedure:
@ PCR consists of a series of repeated
temp. changes, called cycles, each cycle
consists of 2-3 temp. steps.
@ These cycles is preceded by a single temp.
step (called hold) equaling >90°C, and
followed by another hold temp. at the end.
@ The temp. used and the length of time they
are applied in each cycle depend on:
# The enzyme used for DNA synthesis
# The concentration of divalent ions and
Deoxynucleoside triphosphates (dNTPs).
# The melting temp. (Tm) of the primers.

13. Initialization step:
This step consists of heating the reaction to a temp. of 94–96°C and held for 1–9 minutes.
Denaturation step:
@ This step consists of heating the reaction to
94–98°C for 20–30 seconds.
@ It causes DNA melting of the DNA template
yielding single-stranded DNA molecules.
Annealing step:
The reaction temp. is lowered to 50–65°C for 20–40 sec. to allow annealing (attaching) DNA primers to the single stranded DNA template

15. Extension/elongation step:
@ The temp. at this step is 75–80°C which is
required by the Taq DNA polymerase.
@ This DNA polymerase synthesizes a new DNA
strand similar to the DNA template strand.
@ This is done by adding dNTPs to the
template in 5' to 3' direction, then
replaces the 5'-phosphate group of the
dNTPs with the 3'-hydroxyl group at the
end of the chain to extend DNA strand.
@ At each extension step, the DNA amount is
doubled, leading to exponential (geometric)
amplification of the DNA fragment.

16. PCR cycle. (1) Denaturing (2) Annealing (3) Elongation
PCR cycle. (1) Denaturing (2) Annealing (3) Elongation. Blue lines represent DNA template to which primers (red arrows) anneal; extended by DNA polymerase (light green circles); to give shorter DNA products (green lines); which themselves are used as templates as PCR progresses.

17. Final elongation step:
This step is performed at a temp. of 70–74°C for 5–15 minutes to ensure that any remaining single-stranded DNA is fully extended.
Extension/elongation step:
@ This step is performed at 4–15°C for an
indefinite time.
@ It is used for short-term storage of the
reaction.

19. @ To check whether PCR generated good
amplification agarose gel electrophoresis is
employed for size separation of the PCR
products.
@ The size of PCR products is determined by
comparison with a DNA ladder (a molecular
weight marker), which runs on the gel
alongside the PCR products.

20. PCR products after gel electrophoresis.
No amplification in sample 1; Sample 2 & 3 indicate amplification; Positive control & DNA ladder are shown

21. False PCR:
Contamination causes false amplification of DNA products. This is prevented by:
@ Separation of PCR area from areas for
analysis or purification of PCR products.
@ Use of disposable plastic ware
@ Thorough cleaning of the work surface
between reaction steps.
@ Proper primer design techniques by using
electronic PCR
@ Usage of other buffers and polymerase
enzymes.

23. PCR Applications:
@ DNA extraction: From cell genome DNA by
selective amplification of a specific region of
DNA. This is done by hybridization probes
and DNA cloning, to create copies of DNA
fragments.
@ DNA sequencing: To determine unknown
PCR-amplified sequences by the insertion of
a DNA sequence into a genetic material of
another organism.
@ DNA-based phylogeny: To determine the
evolutionary relationships among organisms.

25. @ Amplification and quantification of DNA:
To estimate the amount of a given sequence
present in a sample (gene expression), using
Real-time PCR.
@ Identification of uncultivable or slow-
growing organisms: Such as mycobacteria,
anaerobic bacteria, or viruses.
@ Early diagnosis of infectious diseases: Such
as leukemia and lymphomas; detecting
malignant cells at a high sensitivity.

26. Genes are expressed by being transcribed into RNA, and this RNA transcript is then translated into protein

27. @ Quantification viral load in a patient (HIV)
@ Diagnosis of hereditary diseases
@ Identification of genetic fingerprints. This is
used in forensic sciences and paternity
testing

28. Paternity fingerprints (1) Father. (2) Child. (3) Mother.

29. (2) Real-time PCR: @ Also called quantitative real time PCR (qPCR)
@ It is a laboratory technique based on the
PCR technique that is used to amplify and
quantify a DNA molecule.
@ In a DNA sample, qPCR enables at the same
time both detection and quantification.
@ Its procedure is same as PCR, But in qPCR
the amplified DNA is detected as the
reaction progresses in real time (immediate).
@ qPCR technique can either use non-specific
fluorochromes or hybridization probes

31. Applications of qPCR:
@ Clinical quantification and genotyping
@ Quantification of microbial load in foods or
on vegetables.
@ Rapid detection of cancer, newly emerging
diseases, and genetic abnormalities.
@ Microbial assessment of water quality.
@ Quantitative measure of gene transcription
@ Production of plant seedlings that are free
of pathogens.