Isolation of phaC gene from marine bacteria Paracoccus homiensis strain E33 by magnetic beads subtractive hybridization

Abstract

The isolation of unknown DNA sequences is an important task in molecular biology research. The subtractive hybridization by magnetic beads technique reduces time and cost when the aim of the study is to isolate new genes. We have developed an alternative strategy that combines the subtractive hybridization method with an enrichment microsatellite strategy to isolate a de novo DNA sequence of the phaC gene involved in the polymerization of polyhydroxyalkanoates. A 113-bp fragment was developed as a probe and, based on its melting temperature calculated using the nearestneighbor model, used to isolate the full-length phaC gene (GenBank Accession number HM026759) of the marine bacteria Paracoccus homiensis strain E33, which was isolated from polluted microbial mats. © 2012 Springer-Verlag and the University of Milan.