3-2.3.2.5 Final elongation & Hold:
Final elongation step is occasionally performed for 5–15 minutes at a temperature of 70–74°C after the last PCR cycle to ensure amplification of any remaining single-stranded DNA.
Final hold step at 4°C may be done for short-term storage of the reaction mixture.
After around 30 cycles of denaturation, annealing and extension, there will be over a billion fragments that contain only your target sequence. This will yield a solution of nearly pure target sequence. To check the desired PCR amplification of the target DNA fragment (also sometimes referred to as the amplicon or amplimer), agarose gel electrophoresis is employed for separation of the PCR products based on size. The determination of size(s) of PCR products is performed by comparing with a DNA ladder, which contains DNA fragments of known size, run on the gel along side the PCR products.
3-2.4 Key factor affecting the polymerase chain reaction: Designing of Primers
The specificity of the PCR depends crucially upon the primers. The following factors are important in choosing effective primers.
a) Primers should be 17 to 30 nucleotides in length. However for certain studies the RAPD primer length might be of 10-12 nucleotides.
b) Ideally GC content of the primers is 50% .For primers with a low GC content, it is desirable to choose a long primer so as to avoid a low melting temperature.
c) The basic formula to calculate melting temperature is
Tm = 4°C x (number of G's and C's in the primer) + 2°C x (number of A's and T's in the primer)
Two primers must have a similar Tm value. In case of several primer candidates, we have to choose primers which have the higher Tm value among them.
d) Sequences with long runs (i.e. more than three or four) of a single nucleotide should be avoided.
e) Primers with significant secondary structures (self –hair pin, loop formation) are undesirable.
f) There should be no base complementarities between the two primers.