DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides
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Abstract
The invention relates to genetical modification of DNA polymerase to reduce its innate selective sequence-related discrimination against incorporation of fluorescent dye-labeled ddCTP and ddATP in the enzymatic reaction for preparation of samples for automated florescent dye-labeled terminator DNA sequencing. The modified DNA polymerases are more resistant to heat inactivation and are more effective in dideoxynucleotide incorporation than current DNA polymerases.
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Citations
29 Claims
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1-23. -23. (canceled)
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24. A method for producing a modified form of a DNA polymerase which during DNA sequencing selectively discriminates against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP but does not discriminate against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddTTP and ddGTP, comprising the step of
modifying a DNA polymerase which has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from a strain of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus, so that the modified DNA polymerase includes threonine, praline and leucine at positions 342-344, respectively, and tyrosine at position 422.
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28. A method for producing a modified form of a DNA polymerase which during DNA sequencing selectively discriminates against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP but does not discriminate against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddTTP and ddGTP, comprising the step of
modifying a nucleotide sequence encoding a DNA polymerase which has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from a strain of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus, so that the modified nucleotide sequence encodes threonine, praline and leucine at positions 342-344, respectively, and tyrosine at position 422.
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29-56. -56. (canceled)
Specification