Patent ID: 6015668
Filing Date: 2000-01-18
Classification: C12N,C12Q,C12Y

Abstract:
A method of sequencing a DNA molecule comprising:(a) hybridizing a primer to a first DNA molecule;(b) contacting said first DNA molecule with deoxyribonucleoside triphosphates, a Thermotoga neapolitana DNA polymerase, and a terminator molecule to form a mixture;(c) incubating said mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5' termini; and(d) separating said synthesized DNA molecules by size so that at least a portion of the nucleotide sequence of said first DNA molecule can be determined; wherein:said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; andfurther wherein:said mutant Thermologa neapolitana DNA polymerase has at least one mutation selected from the group consisting of:(1) a first mutation that reduces, substantially reduces or eliminates 3'-5' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'-5' exonuclease domain of said polymerase;(2) a second mutation that reduces, substantially reduces or eliminates 5'-3' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'-3' exonuclease domain of said polymerase; and(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.