Abstract: The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Abstract: The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Abstract: Sequencing methods and methods for synthesizing DNA probes using mutant bacteriophage T4 DNA polymerases which have increased ability to incorporate modified nucleotides for the synthesis of long or short chains of complementary, modified, e.g., fluorophore-labeled DNA. In general, the mutant T4 DNA polymerases retain 3'.fwdarw.5' exonuclease activity; hence, reduction or elimination of 3'.fwdarw.5' exonuclease activity is not a prerequisite for efficient synthesis of a complementary fluorophore-labeled or other modified DNA. In fact, retention of 3'.fwdarw.5' exonuclease activity increases accuracy of DNA replication, because these exonucleases proofread or edit the product of DNA replication.

Abstract: Variant family B DNA polymerases, examples of which have reduced or no 3'.fwdarw.5' exonuclease activity. These variant polymerases have utility as DNA sequencing polymerases.

Abstract: Method for identifying and isolating variant T4 DNA polymerase by isolating and selecting for T4 strains having variant DNA polymerase defective in DNA replication, and at least one additional mutation which corrects or compensates for said defect in DNA replication; identifying the additional mutation(s) and introducing the mutation(s) into T4 phage or T4 DNA polymerase expression vectors.

Abstract: There are provided variant family B DNA polymerases having no 3'.fwdarw.5' exonuclease activity. These variant polymerases have utility as DNA sequencing polymerases. Methods for DNA sequencing with family B DNA polymerases and chain-terminating nucleotides not previously used for sequencing have been developed. The methods disclosed involve the use of family B DNA polymerases not known heretofore to have utility in DNA sequencing, such as variant or wild type forms of phage T4 DNA polymerase or Escherichia coli DNA polymerase II, with novel combinations of deoxynucleotides and chain-terminating nucleotides.