Emphasized-RAPD (e-RAPD): A simple and efficient technique to make RAPD bands clearer

Abstract

The random amplified polymorphic DNA (RAPD) technique has been used widely for purposes such as the construction of linkage maps, QTL analysis, evaluation of genetic diversity, and parentage tests. However, some minor bands of RAPD display a low reproducibility and low reliability. We describe a simple and efficient method for making minor RAPD bands clearer. The first step of this method is to synthesize primers with nucleotides (A, T, G, or C) added to the 3′-end of the original primer sequences. The second step is to perform PCR using them and their combinations with the original primer. The final step is to conduct an electrophoresis analysis. The target bands can be emphasized, and needless background bands can be eliminated. The resulting emphasized RAPD (e-RAPD) bands are clearer and show a higher reproducibility than the original bands. This method has four advantages. (1) The developed e-RAPD bands can be used directly for marker-assisted selection (MAS). (2) The amount of DNA in the target band can be increased and, as a result, conversion to an STS marker, if necessary, is easy. (3) There is a lower risk of missing polymorphisms than with conversion to a sequence-tagged site (STS) marker. (4) The method requires only primer synthesis and PCR, and thus after primer synthesis, the process can be completed within several hours. Easy, low-cost and time-saving detection system is a prerequisite for practical MAS for breeding purposes. For practical MAS, the method presented here using RAPD and conversion to e-RAPD is simpler and easier to apply than using amplified fragment length polymorphism (AFLP) markers or conversion to STS markers.