DNA-CYCLE SEQUENCING OF A COMMON RESTRICTION FRAGMENT OF STAPHYLOCOCCUS-AUREUS BACTERIOPHAGES BY CAPILLARY ELECTROPHORESIS USING REPLACEABLE LINEAR POLYACRYLAMIDE

The nucleotide sequence of a part of a 4.9 kbp common restriction frag ment isolated from Staphylococcus aureus bacteriophage (bacterial viru s) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution a t a temperature of 55 degrees C allowed the reading of more than 650 b ases of sequence in 60 min. The single strand (ss)DNA fragments were p repared by cycle sequencing with fluorescently labeled dideoxy-termina tors on the cloned bacteriophage DNA template. With respect to analysi s speed, sequence read-length, low sample consumption and automation, CE offers a simple, labor-saving and inexpensive procedure for DNA seq uencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read-length. The resu lting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization prob es for identification of Staphylococcus aureus bacteriophages and/or p rophages belonging to the bacteriophage species 3A.