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 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.
Klepárnik
K
K
Institute of Analytical Chemistry, Brno, The Czech Republic. klep@iach.cz
Berka
J
J
Foret
F
F
Doskar
J
J
Kailerová
J
J
Rosypal
S
S
Bocek
P
P
D000180:Acrylic Resins; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D015246:Deoxyribonuclease EcoRI; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013204:Staphylococcus Phages / Q000235:genetics*; D013211:Staphylococcus aureus / Q000821:virology
D000180:Acrylic Resins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005456:Fluorescent Dyes; C016680:polyacrylamide gels; D015246:Deoxyribonuclease EcoRI