Optimization of Colony Polymerase Chain Reaction for the Identification of Actinobacteria by Sequencing of the 16S rDNA

The cell wall composition of actinobacteria is different and more complex compared to other Gram-positive bacteria and varies considerably between genera. Therefore, the isolation of necessary template DNA in the amplification of specific DNA sequences from actinobacteria in appropriate amounts and purity may take a long time and is difficult for polymerase chain reaction (PCR) applications. In this protocol, rapid, simple, and low-cost direct colony PCR methods without prior extraction of genomic DNA as the template for the detection of 16S rDNA sequences in actinobacteria were optimized using 86 different actinobacteria (Streptomyces sp., Micromonospora sp., Nocardia sp., Nocardiopsis sp. Actinomadura sp., Actinopolyspora sp., Saccharopolyspora sp., and Rhodococcus sp. strains). The optimized protocol involved the picking up of microscopic amounts of mycelia with toothpicks and directly transferring them to reaction mixtures (50 μI) which were immediately subjected to PCR. Stable and clear amplification of the 16S rDNA sequence of actinobacteria strains was observed by colony PCR using universal primers of 27F and 1525R. It turned out that the initial denaturation temperature and time are very important in the amplification of specific regions of actinobacteria by colony PCR. The final concentrations of dimethyl sulfoxide (DMSO) and Triton-x100 required for lysis and stability must be added to the PCR as 5% and 0.5%, respectively. In addition, parameters such as the type of commercial DNA polymerase used, the amount of mycelium transferred, and the age of the colony affect the quality of the PCR amplicons.