Geoscience Reference
In-Depth Information
c. Transfer of separated fragments from agarose gel to a
filter by Southern blotting
d. Detection of individual fragments by nucleic acid
hybridisation with a labelled probe(s)
e. Autoradiography
2
.
PCR-based markers.
PCR is a molecular biology tech-
nique for enzymatically replicating (amplifying) small
quantities of DNA without the use of a living organism. It is
used to amplify a short (usually up to 10 kb), well-defined
part of a DNA strand from a single gene or part of a gene.
Since its invention by Kary Mullis in 1983, this technique
enabled the development of various types of PCR-based
techniques, which honoured him to get the Nobel Prize in
1993. However, the basic PCR procedure was described in
1968 by Kleppe and his co-workers in Khorana's group.
The basic protocol for PCR is simple and is as
follows:
a. Double-stranded DNA is denatured at a high tem-
perature (95°C) to form single strands (templates).
b. Short, single strands of DNA (known as primers) bind
at specific annealing temperatures (which vary with
different conditions) to the single-stranded complemen-
tary templates at ends flanking the target sequences.
c. The temperature is raised usually to 72°C for the
DNA polymerase enzyme to catalyse the template-
directed syntheses of new double-stranded DNA
molecules that are identical in sequence to the start-
ing material.
d. The newly synthesized double-stranded DNA target
sequences are denatured at high temperature, and the
cycle is repeated.
Although the basic protocol of PCR is straightforward,
each application requires optimising the various parameters
for the species to be studied. During the early days of PCR
work, the DNA polymerase would need to be added fresh to
the reaction at each temperature cycle, because thermostable
(high-temperature tolerant) DNA polymerases were not com-
mercially available. The discovery of
Taq
DNA polymerase,
the DNA polymerase present in the bacterium
Thermus aquat-
icus
in hot springs, was decisive for the immense utility and
popularity of PCR-based techniques because of its stability at
high temperature, whereas other DNA polymerases became
become denatured. Nowadays, the PCR technology is much
