Abstract
Background: Microsatellite-based PCR has great utility in microbial typing by analysis of band profiles of PCR amplicons. Bacterial DNA extraction methods affect yield and quality of DNA extract, and consequently subsequent molecular-based studies. The study aimed to evaluate impact of bacterial DNA extraction methods on band profiles of microsatellite-based PCR. Methods: Genomic DNA was extracted from Staphylococcus epidermidis ATCC 12228 using QIAamp kit, tween and SDS methods, and submitted to microsatellite-based PCR using (GACA)4 primer. For each extraction method, 4 concentrations were tested: the first crude extracted DNA and other 3 concentrations obtained by 1:1, 1:3, and 1:5 dilutions of the crude samples. Results: Yield and purity of DNA extracted by QIAamp kit were higher than those of other two methods. The PCR amplified all tested DNAs with generation of band profiles ranged from 2 to 11 bands in number, and from 200 bp to 1500 bp in size. Marked inter-method differences were found due to variation in band numbers and sizes. Band patterns obtained for QIAamp kit and tween methods were more robust than for SDS method. Variations of PCR patterns of different DNA concentrations within each method were minimal. Conclusions: Microsatellite-based PCR band profiles vary with different genomic DNA extraction methods for the same bacterial strain. Quality of extracted DNA is more influencing than its concentration. Therefore, it is recommended to unify the extraction method of bacterial DNA for all tested bacterial strains that are submitted to such type of PCR.
Highlights
Polymerase chain reaction (PCR) is a sensitive and specific molecular-based technique used in identification of causative pathogens in infectious diseases
Microsatellite-based polymerase chain reaction (PCR) band profiles vary with different genomic DNA extraction methods for the same bacterial strain
The study results revealed marked diversity in the band patterns of the PCR amplicons of DNAs obtained from the three tested DNA extraction methods for the same bacterial strain
Summary
Polymerase chain reaction (PCR) is a sensitive and specific molecular-based technique used in identification of causative pathogens in infectious diseases. Microsatellite-based PCR utilizes short nucleotide repeats as primer(s) targeting microsatellites which comprise DNA tandem repeats in the target genome [1] It is considered as a simple, rapid, single-step PCR, and proved to have utility in detection, species differentiation, strain identification and genotyping of several bacteria and fungi [2-4]. The key factor used in the interpretation of the results of this PCR variety is the band pattern of the amplified products electrophoresed in agarose gel This pattern depends mainly on polymorphisms in lengths of the target repetitive DNA sequences in tested nucleic acid. Methods: Genomic DNA was extracted from Staphylococcus epidermidis ATCC 12228 using QIAamp kit, tween and SDS methods, and submitted to microsatellite-based PCR using (GACA) primer. Conclusions: Microsatellite-based PCR band profiles vary with different genomic DNA extraction methods for the same bacterial strain.
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