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

118 Dassanayake and Samaranayake
conditions for reproducible PCR (4), because variable or absent PCR products may
result depending on the purity, quantity, or the quality of the DNA templates (5).
The reproducibility of the RAPD technique can also be affected by minor changes in
methodological aspects, such as the differences in the primer-to-template concentration
ratio (see Note 2), variations of primer annealing temperatures, the cation concentration
of PCR buffer, and magnesium ions in the reaction mixture (6, see Note 3). These
parameters can dramatically affect the presence of low-intensity bands as well as the
position and intensity of high-intensity bands. Moreover, some have reported that
different lots of Taq polymerases (see Note 4) and the brand of the thermocycler used
(see Note 5) can also affect the RAPD patterns, especially the low-intensity bands (7).
Furthermore, in some situations, the bands that show equal electrophoretic mobility
may not be homologes, and missing bands may not necessarily reflect homology
because they can be lost by nucleotide substitutions in either the PCR priming sites
or by length mutations. These complications of identity can be resolved either by
sequencing the homologous bands or using a band-specific probe. Such strategies have
been used in several recent studies (8–10).
Our studies are mostly focused on the genomic analysis of the human fungal
pathogen Candida albicans, and the following protocol is based on this experience.
However, the principles guiding RAPD analyses are similar, and the following methods
with minor variations would be applicable in general for many other organisms.
2. Materials
1. Thermocycler.
2. Agarose gel electrophoresis apparatus.
3. Ultraviolet transilluminator, Power supply (200 V and 150 mA).
4. Spectrophometer for determining DNA concentration.
5. Photographic unit that can capture ethidium bromide stained gel (e.g., Polaroid camera
or digital image capture system, such as a CCD camera, a computer, and image analysis
software for clear resolution of the gel image).
6. 10× PCR buffer: 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl 2 , 0.01% (w/v) gelatin,
pH 8.3.
7. 25 mM Magnesium chloride.
8. High-quality sterile, deionized water (more than 10 megaohm/cm) must be used for the
preparation of all reagents and premixes.
9. Deoxynucleotide triphosphate stock solution (dNTP): 2 mM each of dGTP, dATP, dCTP,
and dTTP. Ready-made dNTP (100 mM) solution (obtainable from Sigma, Pharmacia,
Promega, etc.). Make aliquots, preferably 10 mM, and store in –20°C. If dNTP solutions
are made from dry reagents, the pH of the solution should be adjusted to 7.5 with 0.1 M
Tris or 0.1 M NaOH using a pH meter or strip of pH paper.
10. Primers: Lyophilized primers (5′GCGATCCCCA3) should be prepared at 100 µM and 10 µM
concentration with deionized water; Store at –20°C.
11. Taq DNA polymerases: Taq DNA polymerase (5 U/µL; Sigma), Ampli Taq (5 U/µL;
PerkinElmer), Stoffel fragment of Taq DNA polymerase (10 U/µL; Perkin–Elmer), or any
other high-quality Taq polymerase is preferred.
12. Template DNA: 10 to 25 ng/µL stock solution containing good-quality, protein-free,
nonsheared DNA; DNA can be resuspended in high-quality sterile, deionized water or TE
(Tris-EDTA) pH 8.0. RNase (20 ng per 1 ng of DNA) treatment can increase amplification
several-fold. RNase added should be DNase-free.
13. Sterile mineral oil.