LARGE-SCALE, COST-EFFECTIVE SCREENING OF PCR PRODUCTS IN MARKER-ASSISTED SELECTION APPLICATIONS

A simple, PCR-based method has been developed for the rapid genotyping of large numbers of samples. The method involves a alkaline extractio n of DNA from plant tissue using a slight modification of the procedur e of Wang et al. (Nucleic Acids Res 21:4153-4154, 1993). Template DNA is amplified using allele-specific associated primers (ASAPs) which, a t stringent annealing temperatures, generate only a single DNA fragmen t and only in those individuals possessing the appropriate allele. Thi s approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele ar e identified by direct staining of DNA with ethidium bromide. Total te chnician time required for extraction, amplification and detection of 96 samples is about 4 h, and this time requirement can be reduced by a utomation. Excluding labor, cost per sample is less than $0.40. The me thod is tested using the codominant isozyme marker, alcohol dehydrogen ase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).