Biology Reference
In-Depth Information
The STR markers have become the genetic polymorphism of choice in forensic
genetics and PCR is a vital part of the analytical process.
DNA replication: the basis of the PCR
PCR takes advantage of the enzymatic processes of DNA replication. During every
cell cycle the entire DNA content of a cell is duplicated. This copying of DNA can
be replicated outside the cell,
in vitro
, to amplify specific regions of DNA.
The components of PCR
PCR has the following components: template DNA, at least two primers, a ther-
mostable DNA polymerase such as
Ta q
polymerase, magnesium chloride, deoxynu-
cleotide triphosphates and a buffer.
Template DNA
The amount of DNA added to a PCR depends on the sensitivity of the reaction:
for most forensic purposes the PCR is highly optimized so that it will work with
low levels of template. Most commercial kits require between 0.5 ng and 2.5 ng of
extracted DNA for optimum results. This represents between 166 and 833 copies of
the haploid human genome; one copy of the human genome contains approximately
3 pg of DNA. Most forensic profiling can be carried out successfully with fewer
templates - even below 100 pg or 33 copies of the genome [18, 19]; however, the
interpretation of profiles can become more complex as the amount of template DNA
is reduced.
Taq
DNA polymerase
The first PCRs were carried out using a DNA polymerase that was isolated from
Esherichia coli;
in each cycle of the PCR the enzyme was inactivated by the high
temperatures in the denaturation phase and fresh enzyme had to be added [1 - 3].
Fortunately, this is no longer necessary. Scientists were able to isolate the DNA
polymerases from the thermophilic bacteria,
Thermus aquaticus
[20], which was
discovered in the 1960s in the hot springs of Yellowstone National Park, USA.
The
Ta q
polymerase enzyme can tolerate the high temperatures that are involved in
the PCR and works optimally at 72
◦
C-80
◦
C [21]. Using the thermostable enzyme
greatly simplifies the PCR procedure and also increases the specificity, sensitivity and
yield of the reaction [21]. The
Ta q
polymerase enzyme exhibits significant activity at
room temperature that can lead to the creation of non-specific PCR products; adding
the enzyme to a pre-heated 'hot start' reaction reduces the non-specific binding
and again improves the specificity and yield of a PCR [22]. Modifications to the
commonly used
Ta q
polymerase led to the development of the AmpliTaq Gold
