Detection of heterogeneity of 18S rRNA inter-genes and mutation arising during PCR amplification

Direct sequencing revealed sequence heterogeneity among ribosomal RNA gene (rDNA) operons, consisting of 8 base heterogeneous sites on the 18S rDNA of Galactomyces citri-aurantii IFO 10822, and 6 base heterogeneous sites in the same region on the 18S rDNA of G. citri-aurantii IFO 10821. Sequence analysis of the cloned 18S rRNA genes of 14 species (19 strains) of ascomycetous yeast-like fungi detected a total of 32 substitutions between two cloned sequences from each of 10 strains. Eight substitutions came from heterogeneity of G. citri-aurantii IFO 10822, and 24 substitutions were predicted to be due to misincorporation by the Taq DNA polymerase. A low frequency of random substitution, estimated to occur in PCR at approximately 1 in 2690 nucleotides, was detected; and transitions occurred 7 times more frequently than transversions.