2]Detection of point mutations by solid-phase minisequencing

Abstract

Publisher Summary This chapter discusses the detection of point mutations by solid-phase minisequencing. Many mutation types, including large alterations of chromosomal structures, rearrangements, extensions, deletions, or insertions of varying size, have been identified in the human genome. However, 95% of the identified disease-causing mutations are point mutations affecting one or a few nucleotides. Because genetic disorders rarely are caused by a single mutation per disease gene in a population, the ability to detect several mutations simultaneously per sample is of central importance. Consequently there is a need for reliable and technically simple methods for detecting point mutations both in clinical diagnostics and in research laboratories studying human genetic disorders. The chapter presents a convenient method—solid-phase minisequencing—for the detection of single-nucleotide variations, small deletions, or insertions in DNA fragments amplified by the polymerase chain reaction (PCR) and discusses its application for the detection of numerous disease-causing mutations and single-nucleotide polymorphisms in human genes. The major advantage of this method is that its high specificity allows unequivocal identification of any nucleotide variation under the same reaction conditions. Because the assay is carried out in a solid-phase format, it composes simple manipulations in a microtiter well or test tube format without gel- electrophoretic separation steps.