patterns related to meiotic non dysfunction. Different strains of mice have variable CA repeat
lengths and PCR based methods can be used to identify them, thus allowing for specific
genotypes to be assigned. (Lara et al., 2003).
PCR or polymerase chain reaction was originally developed in 1983 by the American
biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his
pioneering work. PCR is used in molecular biology to make many copies of (amplify) small
sections of DNA or a gene.it consist of following steps:
Initialization: This step is only required for DNA polymerases that require heat activation It
consists of heating the reaction chamber to a temperature of 94–96°C
Denaturation: This step is the first regular cycling event and consists of heating the reaction
chamber to 94–98°C This causes DNA melting, or denaturation, of the double-stranded DNA
template by breaking the hydrogen bonds between complementary bases, yielding two single-
stranded DNA molecules.
Annealing: In the next step, the reaction temperature is lowered to 50–65°C. When the
temperature is lowered to enable the DNA primers to attach to the template DNA.
Extension/elongation: The temperature at this step depends on the DNA polymerase used. Taq
polymerase is thermostable it do not denature in high temperature. When the temperature is
raised and the new strand of DNA is made by the Taq polymerase enzyme. The processes of
denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required
to amplify the DNA target to millions of copies. The formula used to calculate the number of
DNA copies formed after a given number of cycles is 2
, where n is the number of cycles. Thus,
a reaction set for 30 cycle’s results in 2
copies of the original double-stranded DNA target
Agarose gel electrophoresisis a method for separation and analysis of macromolecules
(DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in
clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size
independent) and in biochemistry and molecular biology to separate a mixed population
ofDNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge (Kryndushkin et al., 2003).