Aim: Demonstration to amplify a specific DNA fragment by Polymerase Chain Reaction.

Materials Required:
Glasswares: Measuring cylinder, Beaker Reagents: Ethidium bromide (10 mg/ml)
Other requirements: Thermocycler, Electrophoresis apparatus, UV Transilluminator, Vortex
Mixer, Micropipettes, Tips, Adhesive tape, Microwave/ Hotplate/ Burner, Crushed ice
Storage: On receipt of the PCR Teaching Kit, store Control PCR Product, 1 Kb DNA Ladder and
all PCR reagents at -20oC and 6X Gel Loading Buffer should be stored at 2-8oC. Other reagents
can be stored at room temperature (15-25oC).
Theory:
Polymerase Chain Reaction (PCR) is an in vitro method of enzymatic synthesis of specific DNA
fragment developed by Kary Mullis in 1983. It is a very simple technique for characterizing,
analyzing and synthesizing DNA from virtually any living organism (plant, animal, virus,
bacteria). PCR is used to amplify a precise fragment of DNA from a complex mixture of starting
material called as template DNA.

A thermal cycler for PCR

PCR is a very powerful amplification tool so very little DNA is actually required. To copy DNA,
all polymerases require a short sequence of nucleotides to provide a free 3’OH group.
Within cells of most organisms enzymes unwind the duplex and then RNA polymerase adds
priming nucleotides. This priming allows the attachment of DNA polymerase from where
extension can occur. In PCR however, there is a different mechanism of replication. PCR
requires that the flanking sequences of the target DNA be known, so that single stranded
synthetic oligonucleotide primers can be generated. Once these have annealed to the target
DNA the polymerase can synthesize the rest of the chain. Primers can be specific to a
particular sequence, or they can be universal to sequences that are very common within a
DNA molecule allowing for a wide variety of DNA templates. Deoxyadenylate (A),
deoxythymidylate (T), deoxyguanylate (G) and deoxyctidylate (C) are components of the
reaction mixture that are the basic building blocks of DNA required for the synthesis of the
primers and for their extension. The primers and the deoxynucleotides are added to the
mixture in excess.
The most commonly used polymerase in PCR is referred to as Taq-polymerase. This enzyme
was originally found in the 1970's from the thermophilic bacterium Thermus aquaticus.
Before its discovery, PCR was a much longer and costlier process since the thermosensitive
E.coli DNA polymerase used lost its activity during the heating process and fresh enzyme
had to be added at every cycle. Owing that T. aquaticus lives in a hot environment (approx.
75°C), the enzymes that power its internal metabolism have adapted to these conditions
resulting in heat-stability. This polymerase will therefore, retains its activity when the
mixture is heated and can be used for cycle after cycle. A disadvantage of Taq-polymerase is
that it does this without proofreading the newly synthesized DNA. This means that
sometimes the polymerase will include dNTPs that are not complementary to the template
DNA which results in errors in coding. Magnesium ions are cofactors used in PCR specifically
by the polymerase. They affect the annealing of the oligonucleotides to the template DNA
by stabilizing the interaction, and also stabilize the replication complex of the polymerase
with the template primer.

Stages of PCR:
• Denaturation of Target DNA: During this step, the two complementary strands melt
to form single stranded DNA and all enzymatic reactions stop. This is generally
carried out at 92°C – 96°C
• Annealing of Primers : Annealing of primers to each original strand for new strand
synthesis is carried out between 55°C – 65°C
• Extension : : The temperature is again increased to 72°C, ideal for the activity of the
polymerase used, which adds dNTPs complementary to the template at the 3’ end
of the primers.
• These 3 steps are repeated 25-35 times in an automated thermocycler that can heat
and cool the reaction mixture in tubes within a very short time. Each time these
three steps are repeated, the number of copied DNA molecules doubles. Since both
strands are copied during PCR, there is an exponential increase in the number of
copies of the specific DNA fragments, ends of which are defined by 5’ ends of the
primers. The doubling of number of DNA strands corresponding to the target
sequences allows us to estimate the amplification associated with each cycle using
the formula:
Amplification = 2n, where n = Number of cycles
 Fig 1: A PCR amplifies a specific DNA fragment by the incorporation of primers,
dNTPs and Taq polymerase with periodic denaturation and renaturation of the
template DNA.

PROCEDURE :

1) To a PCR tube the following ingredients are added in an order -

Sr. No Ingredients for PCR Volume in
microliters
Molecular Biology Grade
1 33 μl
Water

2 10X Assay Buffer 5 μl

3 Template DNA 2 μl

4 dNTP Mix 5 μl

5 Forward Primer 2 μl

6 Reverse Primer 2 μl

7 Taq DNA Polymerase 1 μl

Total volume 50 μl
 2) The contents were mixed gently and divided equally in 5 PCR tubes.
3) 2 μl of DNA samples were added in the respective tubes.
4) The amplification was carried out in a thermocycler using the following
conditions:
Temperature (ºC) Time
Initial Denaturation
Denaturation
For __
Annealing
Cycles
Extension
Final Extension

5) PCR product was fractionated and shown on agarose gel electrophoresis.

OBSERVATIONS AND RESULTS-

The agarose gel after electrophoresis.

LANE 1:100Bp DNA ladder

LANE 3-8:150bp PCR products
APPLICATIONS OF PCR:

1)CLINICAL USES-
• DIAGNOSING GENETIC DISEASE
• MONITORING CANCER THERAPY
• SCREENING GENES FOR MUTATION
• PRENATAL DIAGNOSIS
2)NON-CLINICAL USES-
• SCREENING CLONES
• SEQUENCING
• CLONING GENES
• STUDYING MOLECULAR EVOLUTION

Precautions:
1. Minimize damage to template DNA by avoiding vortexing or vigourous mixing
2. Store the kit at -20oC and avoid repeated freeze thaw. Also keep all the materials in
ice while performing the experiment
3. Ensure proper functioning of Thermocycler. Run positive control with every reaction
4. Mix the reaction mixture using a micropipette, avoid air bubble
5. Work in sterile conditions to avoid contamination. Avoid vigorous mixing of the DNA
samples