General Genetics
1.Introduce the students to the preparation of
the PCR reaction.
2.Examine the PCR products on agarose gel
electrophoresis.
3.Explain some of PCR applications.


Kary mullis received a Nobel Prize in
chemistry in 1993, for his invention of the
polymerase chain reaction (PCR).
The
process,
which
Kary
Mullis
conceptualized in 1983, is hailed as one of the
monumental scientific techniques of the
twentieth century.
 PCR is used to amplify specific regions
of a DNA strand (the DNA target) in
vitro.
 This can be a single gene, a part of a
gene, or a non-coding sequence.
 Most PCR methods typically amplify DNA
fragments of up to 10 kilo base pairs (kb),
although some techniques allow for
amplification of fragments up to 40 kb in size.

1.
2.
3.
4.
5.
The solution must include:
The template DNA
A thermostable DNA polymerase
Two oligonucleotide primers
Deoxynucleotide triphosphates (dNTPs)
Reaction buffer (Tris, ammonium ions (and/or
potassium ions), magnesium ions, bovine serum
albumin).
This components must be mixed together in a solution
with a total volume of between 25 and 100 microliters.

Once assembled, the reaction is placed in a thermal cycler,
an instrument that subjects the reaction to a series of
different temperatures for set amounts of time.

This series of temperature and time adjustments is referred
to as one cycle of amplification.

Each PCR cycle theoretically doubles the amount of
targeted sequence (amplicon) in the reaction.

At 30 cycles there are 1,073,741,764 target copies (~1×109).

Each cycle of PCR includes steps for DNA
template :
1. Denaturation :
94°C
15 sec_2 min
2. Primer annealing: 40–60°C 15 sec_60 sec
3. Primer extension: 70–74°C 1–2 minutes

Denatures the target DNA by heating it to
94°C for 15 seconds to 2 minutes.

The two intertwined strands of DNA separate
from one another, producing the necessary
single-stranded DNA template for replication
by the thermostable DNA polymerase.
Heating

The temperature is reduced to approximately
40–60°C.

At this temperature, the oligonucleotide
primers can form stable associations (anneal)
with the denatured target DNA and serve as
primers for the DNA polymerase.

This step lasts approximately 15–60 seconds.

The synthesis of new DNA begins as the
reaction temperature is raised to the
optimum for the DNA polymerase.

For most thermostable DNA polymerases,
this temperature is in the range of 70–74°C.

The extension step lasts approximately 1–2
minutes.

The next cycle begins with a return to 94°C
for denaturation.

After 30–40 cycles, the amplified product
may then be analyzed for size, quantity,
sequence, etc., or used in further
experimental procedures.



Is a species of bacterium that can tolerate high
temperatures one of several thermophilic bacteria.
It is the source of the heat-resistant enzyme Taq DNA
Polymerase, one of the most important enzymes in
molecular biology because of its use in the PCR (DNA
amplification technique).
Taq produces an enzyme called DNA polymerase, that
amplifies the DNA from the primers by the polymerase
chain reaction, in the presence of Mg.