Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing

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, followed by affinity-capture techniques for convenient and efficient purification of the PCR fragments for sequencing. Furthermore, the cyclic Sanger sequencing reactions are easy to automate with laboratory robots, and instruments have been developed for automatic online 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 is needed, as in DNA diagnosis or in the analysis of sequence variation for genetic, evolutionary or epidemiological studies.