Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase.
We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA. The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel. Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms. Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity. These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers.
Kricker
M C
MC
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Tindall
K R
KR
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D013604:T-Phages / Q000235:genetics*; D019914:Taq Polymerase; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology
D004279:DNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase