DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides

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.

1 - 23 . (canceled)  
     
     
         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.    
     
     
         25 . The method according to  claim 24 , wherein the DNA polymerase has proofreading 3′-5′ exonuclease activity during DNA sequencing of a DNA strand from a template, such that the DNA polymerase functions to excise mismatched nucleotides from the 3′ terminus of the DNA strand at a faster rate than the rate at which the DNA polymerase functions to remove nucleotides matched correctly with nucleotides of the template.  
     
     
         26 . The method according to  claim 24 , wherein the DNA polymerase has the amino acid sequence of SEQ ID NO:4.  
     
     
         27 . The method according to  claim 24 , wherein the DNA polymerase is encoded by a DNA segment having the nucleotide sequence of SEQ ID NO:3.  
     
     
         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.    
     
     
         29 - 56 . (canceled)