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

RAPD Fingerprinting 121
Fig. 1. RAPD fingerprinting of 15 clinical Candida parapsilosis isolates (P1 to P10 superficial
and P11 to P15 systemic) with primers RSD12 (5′ GCA TAT CAA TAA GCG CAG GAA
AAG 3′) (A) and RSD6 (5′ GCG ATC CCC A 3′) (B) obtained after electrophoretic separation
on 1.2% agarose gel. M, PCR marker (Sigma). Sizes of bands indicate the number of base
pairs (18).
annealing temperatures, cycle number and time duration of denaturation, annealing and
primer extension period, and the type of thermal cycler used (4). Different varieties of
thermocyclers, with intrinsic inhomogeneneities in rates of cooling and heating, can elicit
incongruent fingerprints despite identical program settings and or reaction components.
Similarly, subtle changes in the temperature within the same heat block can alter the
mode of amplification. Annealing temperature also impacts the quality of the fingerprints
produced and their reproducibility. It is noteworthy that the lifetime of a polymerizing agent
can be extended considerably by reducing the duration of the denaturation temperature.
6. Template: It is well known that the purity of the template is critical for producing
reproducible fingerprinting patterns; thus, DNA devoid of proteins and RNA must be used
at all times. For instance, impurities, such as phenol remnants in DNA, can be removed by
repeated washing in 70% ethanol. Also DNA isolation methods that lead to degradation
of DNA and inhibit the activity of DNA polymerase should be avoided. Furthermore,
the RAPD technique cannot be reproducibly used to amplify DNA beyond a minimal
threshold (less than 5 ng) or at the other extreme, a very high concentration of template
DNA (higher than 1 µg). Such attempts invariably lead to production of either “smears”
or poor resolution of the amplicons. In general, 50 to 100 ng of DNA can be used for
50 µL of PCR reaction mixture (17).