A GENE FROM THE BACTERIUM RHODOTHERMUS MARINUS, CODING FOR THE AMINO ACID SEQUENCE OF DNA-LIGASE, PRODUCED IN ESCHERICHIA COLI OR SOME OTHER SUITABLE HOST ORGANISM

摘要

This invention provides a new DNA ligase enzyme, derived from the thermophilic bacterium Rhodothermus marinus, active at temperatures between 5-75 °C with half life of 7 min. at 90 °C. The invention constitutes the nucleotide sequence of the gene, coding for the enzyme, its expression in a productive host, purification of the protein to near homogeneity and the use of the enzyme in cycle sequencing. Unique properties of this enzyme make it suitable for use in the SPEL-6 method of primer walking (sequential primer elongation by ligation of 6-mers) initially described by Szybalski (5) and computer-controlled automatic sequencing of long DNA sequences.