DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary...

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 degrees 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 fluorescently 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.