Direct amplification by RAPDs of fresh leaf disc tissue
--Emily Chin and Stephen Smith

Most of the RAPD analyses done to date require a carefully calculated concentration of extracted genomic DNA as the target source. Using three inbred lines of maize, we were able to directly amplify and generate the same DNA profiles from fresh leaf tissue as were obtained using extracts made from lyophilized leaf tissue from two week old seedlings. Leaf discs collected from three-four week plants were analyzed using both conventional PCR and with RAPD. Leaf discs were immersed directly into the amplification cocktail without additional attempts at extracting DNA. DNA of the same inbred lines extracted from lyophilized leaf flour was also amplified under the same PCR or RAPD conditions in order to compare banding profiles obtained directly from leaf discs versus the more conventional source of DNA.

Amplifications obtained via "classical" PCR using a pair of primers gave clear and repeatable banding profiles regardless of whether the DNA had been extracted from lyophilized flour or whether the reaction occurred instead in the presence of an immersed fresh leaf disc. With RAPD, banding profiles obtained from reactions that had DNA from lyophilized leaf flour also gave clear profiles. However, with fresh leaf discs as the source of DNA, the RAPD method did not result in any visually obvious bands.

For each of the samples that had utilized a fresh leaf disc as the potential source of target DNA, an aliquot was taken following one round of amplification to then initiate a sequential and second round of amplification. Following two rounds of amplification for the PCR reactions, there were several bands that were either not present or only faintly present following the first round of amplification. However, with RAPD, the second round of amplification resulted in clear and distinct banding profiles that were very similar to those obtained from a single round of amplification when DNA extracted from lyophilized leaf flour had been the target source. These results showed that the binding of primers to target DNA in a RAPD reaction could be less efficient than with PCR. However, once a primer-target is established, then amplification can occur with high fidelity. Fresh leaf discs, therefore, could be used to generate RAPDs but two sequential rounds of amplification would then be necessary. 


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