DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide

Abstract

The nucleotide sequence of a part of a 4.9 kbp common restriction fragment isolated from Staphylococcus aureus bacteriophage (bacterial virus) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution at a temperature of 55°C allowed the reading of more than 650 bases of sequence in 60 min. The single strand (ss)DNA fragments were prepared by cycle sequencing with fluo‐rescently labeled dideoxy‐terminators on the cloned bacteriophage DNA template. With respect to analysis speed, sequence read‐length, low sample consumption and automation, CE offers a simple, labor‐saving and inexpensive procedure for DNA sequencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read‐length. The resulting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization probes for identification of Staphylococcus aureus bacteriophages and/or prophages belonging to the bacteriophage species 3A.