Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing

Abstract

The Sanger dideoxy-nucleotide sequencing method has been simplified by a number of methodological improvements, such as the use of the polymerase chain reaction (PCR) technique for generating DNA templates in sufficient quantities for sequencing, the use of affinity-capture techniques for convenient and efficient purification of the PCR fragments for sequencing, the development of laboratory robots for carrying out the sequencing reactions, and the development of instruments for automatic on-line analysis of fluorescent products of the sequencing reactions. Despite these technical improvements, the requirement for gel electrophoretic separation remains an obstacle when sequence analysis of large numbers of samples are needed, as in DNA diagnosis, or in the analysis of sequence variation for genetic, evolutionary, or epidemiological studies.