RAPD technology developed by Williams, Tingey, and Rafalski (Nucleic Acids Res. 18:6531-6535, 1990) is being used as a tool to generate molecular markers. We have successfully applied this technology to maize and are able to obtain clear reproducible data. To optimize clarity and reproducibility of profiles, we investigated the effects of different cycling parameters in order to develop a set of routinely applicable and reliable protocols for RAPD in maize. DNA is extracted (Jhingan, Meth. Mol. Biol. 3:15-22) from lyophilized leaf flour taken from 2 week old seedlings. Details of the protocol are given below.
The most expensive component of the reaction is the polymerase enzyme. Using varying amounts of Taq, ranging from 0.5 units to 2.0 units per reaction (with 20ng genomic DNA), and annealing temperatures of either 35 or 37 C, we established an optimum concentration of 0.8 units of Taq per a 25µl reaction. Too much Taq was undesirable because it resulted in high background presumably caused by non-specific binding during annealment. Too little Taq did not allow any visibly detectable amplification.
We have used annealing temperatures of 31, 33, 35, 36, 37, 39, and 41 C each with one unit of Taq for 45 cycles; no differences for the moderate to brightly fluorescing bands (with EtBr) could be seen. We also found no differences for these bands between annealment at 35 C and Touchdown RAPD from 42 C ramping down to 36 C.
Another component of the reaction mix we investigated was the concentration of dNTP mix. A high concentration of readily available dNTP theoretically should allow for more efficient incorporation of bases during primer extension. We tried 0.3, 0.6, 0.9, and 1.2mM dNTP per reaction. Our results indicated that a concentration of 0.6 dNTP was sufficient to generate clear and reproducible profiles.
Since the number of cycles used in the amplification process significantly determines the total duration of profile generation via PCR, we established the minimum number of cycles that would be necessary to generate clear and reproducible profiles. After trying amplification with 25, 30, 35, 40, and 45 cycles we found that 40 cycles could be used most reliably and efficiently.
Our current protocol for RAPDs in maize is as follows:
1) DNA extraction (Jhingan, Meth. Mol. Cell Biol. 3:15-22);
2) Reaction mix: 2.5µl 10X buffer, 0.6µl 10mM dNTP, 0.8 unit Taq polymerase, 5.0pM primer (10mer), 20.0ng DNA template (buffer is 67mM Tris-HCl pH 8.8, 16.6mM (NH4)2SO4, 6.7mM MgCl2, 10mM DTT, 0.01% NP-40, 0.01% Tween-20);
3) Cycling profile is
a) initial denaturation of DNA into single strands by 94 C 3.00 min;
b) 40 cycles of:
i) 94 C 1.00 min;
ii) annealing at 37 C 1.00 min;
iii) extension at 72 C 2.00 min;
c) 72 C 7.00 min, then hold at 4 C until gel loading or store frozen prior to gel loading;
4) Electrophoretic conditions - run on 2% NuSieve 3:1 agarose gel after adding loading dye to each sample.
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