Abstract
We attempted colony PCR without prior extraction of DNA as the template for rapid and simple detection of specific DNA sequences in actinomycetes. Microscopic amounts of mycelia were picked up with toothpicks and directly transferred to PCR mixtures (20 μl) which were immediately subjected to heating (95°C, 3 min) followed by 30 cycles of PCR (95°C, 30 sec → 60°C, 30 sec → 72°C, 60 sec). Specific and clear amplification of the target region (518 bp) of an aminoglyocoside acetyltransferase (AAC) gene, kan, coding for an AAC(3) in Streptomyces griseus strains was observed by colony PCR using originally designed 20mer oligonucleotide primers (kanl and kan2). Stable amplification was not obtainable by the addition of spore-bearing aerial mycelia or visible amounts of mycelia. Good amplification of 16S rDNA sequence (1.5 kb) was also detected by using known primers of 9F (19mer) and 1541R (17mer). Employing a higher annealing temperature (65°C) provided good amplification with better specificity of the target sequences of kan and 16S rDNA. Findings confirm that colony PCR will definitely be useful for characterization and manipulation of genes in actinomycetes.
