Stéphane Mabic

Impact of purified water quality on molecular biology experiments.

Abstract Purified water is a reagent used in a variety of molecular biology experiments, for sample and media preparation, in mobile phases of liquid chromatography techniques, and in rinsing steps. The combination of several technologies in water purification systems allows delivering high-purity water adapted to each application and technique. Through a series of examples, the importance of water quality on biotechnology experiments, such as single nucleotide polymorphism (SNP) analysis by denaturating HPLC, RNA preparation and PCR, is presented. Results obtained on DNA mutation and single nucleotide polymorphism analysis using the denaturating HPLC (DHPLC) technique highlight the benefits of organic removal by UV photooxidation process. Comparative gel electrophoresis data show that ultrafiltration is as efficient as diethylpyrocarbonate (DEPC) treatment for suppressing RNase activity in water. Gel electrophoresis and densitometry measurement also point out the benefits of ultrafiltration to carry out reverse transcriptase-polymerase chain reaction quantitatively.

Utilizing ultrafiltration to remove alkaline phosphatase from clinical analyzer water.

Abstract Alkaline phosphatase (ALP) conjugated to antibodies is often used in enzyme immunoassays (EIAs). These assays are notably sensitive to experimental conditions. A possible source of interference is bacterial ALP, which is released when bacterial contamination occurs in clinical analyzers. Preliminary experiments led to the selection of a detection kit, ALP source, and specific types of tubes for collecting water samples and performing assays. The release of ALP from various strains of bacteria identified in pure water was demonstrated (10–30×106cfu/mL released 6–10μU/μL). It was shown that ultrafiltration is totally efficient in removing ALP from water, while residual ALP activity (2.21μU/μL after filtration of an ALP solution of 6.22μU/μL) was observed after filtration using a 0.22-μm filter.