Rapid detection of a single-base-pair mutation in phytoplasmal conserved genes by DNA heteroduplex mobility assay (HMA)

Abstract

This paper reports, for the first time, the detection of a single-base-pair mutation in a 16S rDNA fragment derived from a phytoplasma isolate by a DNA heteroduplex mobility assay (HMA). The 16S rDNA fragment was amplified from aster yellows phytoplasma isolate (AY27) by polymerase chain reaction (PCR) and digested by the restriction enzyme AluI. Following the cloning of the partial 16S rDNA fragment into a plasmid vector, the cloned fragment was subjected to mutation in vitro to yield three sets of mutants containing one-, two-, or three-base-pair substitutions. The mutated 16S rDNA fragments were amplified by PCR and analyzed by HMA. The results indicated that the DNA heteroduplexes (529bp) containing at least two-base-pair difference, i.e. a 0.4% level of divergence, were separated from DNA homoduplexes in 5% polyacrylamide gel under nondenaturing conditions. A single-base-pair substitution in a 529bp DNA fragment was differentiable by HMA when the DNA fragment containing more than two-base-pair substitutions was used as a reference.