Direct assessment of viral diversity in soils by random PCR amplification of polymorphic DNA.

Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.

ISSR: a reliable and cost-effective technique for detection of DNA polymorphism.

With the emergence of more and more molecular markers as useful tools in plethora of population genetic and phylogenetic studies, choice of marker system for a particular study has become mind boggling. These marker systems differ in their advantages and disadvantages, so it is imperative to keep in mind all the pros and cons of the technique while selecting one for the problem to be addressed.Here, we have shed light on the ISSR (intersimple sequence repeat) technique, as a marker of choice if one wants to go for properties such as reliability, simplicity, cost effectiveness, and speed, in addition to assessing genetic diversity between closely related individuals. We have outlined here the whole methodology of this technique with an example of Tribulus terrestris as case study.

[Evaluation of three methods for genotyping of nosocomial methicillin resistant Staphylococcus aureus isolates]. / Nozokomiyal metisiline dirençli Staphylococcus aureus izolatlarinin genotiplendirilmesinde üç ayri yöntemin incelenmesi.

Plasmid profile analysis (PPA), random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) are the three most valuable epidemiological tools for genotyping of methicillin resistant Staphylococcus aureus (MRSA) strains. Aim of this study was to evaluate these three methods in respect to their cost, reproducibility, and discriminatory power. Eighty one nosocomial MRSA isolates with unknown genetic and epidemiological relatedness from Training Hospital of Gulhane Military Medical School, were genotyped by PPA, RAPD, and PFGE methods. All isolates (100%) were typed by RAPD and PFGE, however, eight (9.9%) isolates could not be typed by PPA since they lacked plasmid DNA. Reproducibilities of all the three methods were found to be 100 percent. Discriminatory powers of PPA, RAPD and PFGE methods were calculated as 48.6%, 61.1% and 80.1%, respectively. In conclusion, out of the three methods tested, PFGE allowed the most effective discrimination of MRSA strains. However, PFGE was more time consuming and technically demanding, and required use of specialized and expensive equipment. Although PPA and RAPD were less discriminatory than PFGE, these methods were technically simple, rapid and cheaper. When PPA and RAPD were used in combination, they had equal discriminatory power to PFGE. Thus, it should be emphasized that PPA and RAPD methods could be preferred for initial screening purposes while PFGE should be used as a confirmatory test in genotyping of MRSA isolates.

Use of random amplified polymorphic DNA for rapid molecular subtyping of Corynebacterium diphtheriae.

A total of 210 Corynebacterium diphtheriae strains isolated worldwide were assayed by random amplified polymorphic DNA (RAPD) assay. RAPD was as discriminating as standard ribotyping, and in some cases, even further differentiation was obtained. RAPD can rapidly aid clinical and molecular epidemic studies in a simple and cost-effective manner.