John M. S. Bartlett.pdf - Bio-Nica.info

120 Dassanayake and Samaranayake
5. Electrophorese amplified products under 5 to 10 V/cm for 2 to 2.5 h. When the bromophenol
blue travels three quarters of the length of the gel, visualize the gel under ultraviolet
light and photograph.
4. Notes
1. Annealing temperature: This is an important parameter that needs optimization in RAPD
and depends on primer length and sequence. The melting temperature (Tm) of a primer
is proportional to both its length and the G + C content. Tm for primer template can be
determined using the formula given below.
Tm = [(number of A+T) × 2°C + (number of G + C) × 4°C]
However, optimal annealing temperature for a primer should be adjusted empirically.
Generally, in RAPD, the first few cycles are performed at a low annealing temperature
(`5°C below the calculated Tm) and subsequent cycles are performed at a high annealing
temperature (`5°C above the calculated Tm).
2. Primer-template ratio: This is one of the important variables in the amplification reaction.
Generally, moderate primer:template mass ratios ranging from 0.5 to 5000 are used.
3. Ionic composition: The concentration of ionic components is critical for RAPD. Of
these, magnesium is important because different thermostable polymerases have different
affinities for magnesium. Generally, the higher the concentration of the magnesium ions,
the lower is the specificity, and vice versa. In our hands reproducible fingerprints for
C. albicans isolates were obtained with magnesium ion concentrations of 2.5 mM. It is noteworthy
that when DNA is dissolved in TE buffer, the magnesium ion concentration has to be
increased (~3 mM) to obtain reproducible patterns. This is probably caused by the chelation
of magnesium ions by EDTA, which lowers the effective ionic concentration in the reaction
mixture. Further, Weaver et al. (11) reported that excess primer and template DNA could
also modulate the activity of magnesium by sequestrating free magnesium ions and thus
dampening the amplification reaction. The dNTP concentration also has a direct effect
on the magnesium ion concentration in the reaction mixture as a result of the interaction
between the mononucleotide and magnesium. Thus, a higher concentration of magnesium
ions is necessary for amplifications with a higher concentration of dNTPs (12). On the
contrary, high magnesium ion concentrations can lead to primer–template mismatching
and thus decrease amplification stringency. Furthermore, magnesium ions can tightly bond
with the sugar backbone of nucleotides and nucleic acids and therefore variation in the
magnesium concentration has strong and complex effects on nucleic acid interactions.
4. DNA polymerase: The activity of polymerases is highly variable (13) and therefore, subtle
differences in the specificity of these enzymes can influence the fingerprint profiles, and the
multiplex ratio (14,15). The polymerase activity is regulated to a great degree by the buffer
components and, thus, a recommended buffer has to be used for a particular polymerase.
We have observed that the variations in the combinations of buffers and polymerases lead
to inadequately resolved and incomplete fingerprints. Highly variable results are obtained
in particular when different eubacterial DNA polymerases are used in the RAPD technique.
On the contrary, Thermal aquatics Stoffel fragment is a truncated DNA polymerase that
has wide magnesium tolerance and thermal stability and produces well-defined low
molecular weight products (less than 500 bp) in general. RAPD with truncated DNA
polymerases are known to produce good yields. Generally, Taq polymerase concentrations
of 1 to 1.25 U/50 µL-reaction is used in RAPD. However, the reaction mixture of more
than 2 U/µL can generate nonspecific products (16).
5. Thermal cycling parameters: These are of critical importance for optimization of the
RAPD reaction (17). Thermal cycling parameters include template denaturation and