Quantitative Properties of Random Amplified Polymorphic DNA (RAPD)

Abstract

Random Amplified Polymorphic DNA analysis (RAPD) is a methodology that has been used as a tool for monitoring microbial communities. To be useful in this application RAPD, and any other methodology, must show properties that allows for the detection of quantitative changes in composition of the microbiota. Therefore, the objective of this study was to establish whether RAPD possesses such properties. The strategy was to use genomic DNA, extracted from a set of tertiary bacterial mixtures defined according to an experimental mixture design, and containing varying proportions of Escherichia coli, Bacillus subtilis, and Pseudomonas CF600. RAPD-PCR was performed on the mixed DNA extracts and the amplified DNA fragments were separated on sequencing gels to produce genomic fingerprints that were digitized and modeled by Partial Least Squares regression (PLS). Significant predictions were obtained using an external test set for validation, with Root Mean Square Error of Predictions (RMSEP) of 0.21, 0.19 and 0.20 for the proportion of E. coli, B. subtilis and Pseudomonas CF600 respectively. Taken together, the results showed that RAPD patterns quantitatively represented the initial mixture proportions. Therefore, the view that RAPD could be useful for whole microbial community monitoring was strengthened.