Collect. Czech. Chem. Commun. 1997, 62, 1136-1142

Sequencing Strategy for a Protein-Linked Genome: Spontaneous Reversions of Ochre Triplet in the Phage ϕ29 Gene 17

Vladimír Fučíka, Vladimír Chaloupeckýb, Jaroslav Berana and Jiří Jonáka

a Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 166 37 Prague 6, Czech Republic
b Institute of Medical Microbiology, 1st Faculty of Medicine, Charles University, 128 06 Prague 2, Czech Republic


A direct method using T7 gene 6 exonuclease to prepare single-stranded templates was adapted for the sequencing of the phage ϕ29 genome carrying a covalently linked terminal protein at the 5' ends of both DNA chains. The terminal protein was found to block completely the action of the exonuclease. The treatment of the phage DNA by proteinase K did not restore the accessibility of the 5' ends to the exonuclease to the full extent but only partially. The digestion of proteinase K-treated terminal fragments of the genome by 5' exonuclease could proceed now from both 5' ends, however, at different rates. The delay of exonucleolytic digestion apparently due to residual amino acid(s) was calculated to be about 800 nucleotides. The relative rates of exonucleolytic splitting of both chains were of decisive significance for the choice of suitable primers for the sequencing of desired regions. In all Sus+ revertants sequenced by the described method the TAA(ochre) codon in the gene 17 was found changed either to the original CAA(Gln) or to TAT(Tyr) with the same frequencies.

Keywords: Bacteriophage fi29; Spontaneous mutations; Terminal protein; Proteinase K; T7 gene 6 exonuclease.