Patent ID: 6365350
Filing Date: 2002-04-02
Classification: C12Q

Abstract:
A method for determining DNA nucleotide sequences comprisingproviding ribonucleoside 5â€²-triphosphates, wherein said ribonucleoside triphosphates are selected from the group consisting of ATP, GTP, CTP, UTP, and derivatives thereof, reacting said ribonucleoside 5â€² triphosphates with one or more kinds of 3â€²-dNTP derivatives in the presence of an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase to obtain a nucleic acid transcription product, wherein said 3â€²-dNTP derivatives are 3â€²-deoxyribonucleoside 5â€²-triphosphates selected from the group consisting of 3â€²-dATP, 3â€²-dGTP, 3â€²-dCTP, 3â€²-dUTP, and derivatives thereof, separating the resulting nucleic acid transcription product, and determining a nucleic acid sequence from the resulting separated fraction, wherein the RNA polymerase is a mutant promoter-specific RNA polymerase selected from the group consisting of F644Y, L665P, F667Y, F644Y/L665P, F644Y/F667Y, L665P/F667Y and F644Y/L665P/F667Y mutants of an RNA polymerase derived from T7 phage, said mutant RNA polymerase having an enhanced ability for incorporating the 3â€²-dNTP derivatives in comparison with the corresponding wild type RNA polymerase.