Impact of Staphylococci DNA extraction methodology on microsatellite-based PCR banding profile | ||||
| Microbes and Infectious Diseases | ||||
| Article 13, Volume 2, Issue 3, August 2021, Page 476-484 PDF (325.92 K) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/mid.2021.77774.1160 | ||||
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| Author | ||||
Atef Shehata  
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| Department of Microbiology and Immunology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt | ||||
| 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. | ||||
| Keywords | ||||
| DNA extraction; Microsatellite-based PCR; (GACA)4 primer; DNA quality; Banding profile | ||||
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