Influence of Experimental Conditions on the Reproducibility of RAPD-PCR Identification of Legumes and Cereals

Abstract

The experimental conditions for species identification of food legumes and cereals by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) were studied with two legumes (soybeans and lentils) and two cereals (wheat and rye). Eleven isolation protocols using commercial kits with gel cartridges or membrane columns, extraction with chloroform–organic solvent mixtures and different lysis buffers, six commercial 10-mer primers and pre-mixed dried PCR beads as well as Taq DNA polymerase were tested. DNA templates obtained by extracting defatted seed meal with SDS-containing buffer and purified with chloroform:isoamyl alcohol treatment as well as those isolated with the aid of a commercial membrane column kit resulted in distinct and reproducible amplicon patterns after amplification. Only small differences were observed between the six 10-mer primers and both RAPD-PCR beads and Taq DNA polymerase yielded interpretable amplicon patterns. For cereals, Taq DNA polymerase and each of the two isolation methods can be recommended. For legumes, beads are slightly favoured and the two isolation methods can be used. The unexpected lack in amplicon formation which was sometimes observed suggests that all analyses should be run in duplicate.