The Application of Ooubled Haploid Plants to Population ... - MSpace

Application of RAPOs
PCR conditions can be manipulated to influence the RAPD banding pattern.
These conditions include the temperatures controlled by the thermal cyding device
responsible for PCR automation. This device acts by heating matt black metal surfaces
followed by air cooling. Optimum temperatures at different stages of the PCR reaction
Vary based on the primer and genomic DNA being used. The first action of the themal
cycier is to dissociate the double sûanded template DNA with heat in the presence of
primers, a thermostable DNA polymerase, dNTPs, MgCI, and a PCR buffer. The
primers are then annealed to the template DNA by reduction of the temperature to 37 -
65"C, depending on the melting temperature (Tm) of the primers. The primers are then
extended, aided by a thermostable DNA polymerase which adds the dNTPs. These
steps are repeated for ai least 20 cycles. The number of cycles and the duration of
each step influence the number, specificity and reproducibility of the RAPD bands. It is
important to optimize the number of PCR cycles to avoid amplifying background
products which rnay amplify preferentially. The last extension time is usually increased
by several minutes to complete the synthesis of al1 strands (Newton and Graham,
1994).
PCR conditions may be varied in a number of ways. Primers of 20 - 30
nucleotides allow reasonably high Tm which minirnizes the amplifcation of non-specific
products. It is desirable for primer sequences to have at least 50 % guanine and
cytosine and avoid repetitive sequences to minimize amplification of primerd imers and