Chapter 5 - General Procedures

Abstract

This chapter discusses the classical molecular biology methods that are routinely used in clinical molecular diagnostic laboratories, either as part of patient testing procedures or as quality control and/or quality assurance measures. These procedures may also serve as building blocks of more sophisticated molecular diagnostic procedures. Although important molecular biology methods such as plasmid construct preparation, bacterial transformation, or transfection of mammalian cells are rarely if ever used in a molecular diagnostic laboratory, others such as gel electrophoresis, polymerase chain reaction and ultraviolet (UV) spectrometry are commonly used. As the most commonly used analytical procedure in a molecular pathology laboratory, gel electrophoresis allows visualizing patient nucleic acid specimens or their assay products directly. Qualitative as well as quantitative information can be obtained from this procedure. Oftentimes, it is part of an assay procedure either as a quality control measure or as a detection method. Dependent on application needs, different gels or gel formats, such as agarose versus polyacrylamide, or slab gel versus capillary gel, can be used. A standard agarose gel apparatus is also referred to as a submarine apparatus, where a tray containing solidified agarose gel is submerged in buffer solution during electrophoresis. A gel tray is usually placed in a gel apparatus filled with running buffer before sample loading.