Primers used with the RAPD procedure are assumed to perhaps have only a partial homology to the target sequences. However, we have evidence to show that, at least in maize, this homology appears rather to be quite precise.
Firstly, it is usual to employ a less stringent annealing temperature (e.g. 35 C) in RAPD analyses in order to allow for potential mismatches between primer and target sequences. However, we have used Touchdown RAPD with annealing temperature initially at 42 C and reducing to 31 C. This procedure resulted in the same amplification profile as when compared to the profiles for samples run under annealment at 35 C alone.
Secondly, we have performed a series of RAPD amplifications using different annealing temperatures. The annealing temperatures were 31, 33, 35, 36, 37, 39, and 41 C. The amplification products were all run on 2% agarose gels and no differences for the moderately to brightly fluorescing bands could be noted. This range of annealing temperatures would be expected to encompass a very wide range of stringencies for a 10mer.
Thirdly, we synthesized a series of 11 nucleotides each 10 bases in length. Pairs of these primers differed by only a single nucleotide from each other, whilst maintaining the purine pyrimidine ratio. These primers were used to amplify, via the RAPD procedure, nine maize inbred lines that collectively encompassed a broad range of Corn Belt genetic diversity. Observation of the RAPD profiles showed that varying a single base anywhere on the primer, irrespective of whether it was toward the 3' or 5' end, resulted in completely different banding profiles when applied to the same corn inbred line. One might have expected, if annealment occurred with less than at least a fairly complete homology between primer and target sequence, that substitution of single bases in the primer would sometimes not be consequential with respect to the outcome of the amplified products. We never found this to be the case, thereby demonstrating a degree of specificity of primers in respect to the target sequence.
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