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AssigneeVisible Genetics Inc.
InventorsJames Leushner, Jean-Michel Lacroix, May Hui, James M. Dunn, Marina T. Larson
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Sequencing of a selected region of a target nucleic acid polymer in a natural abundance DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The reaction mixture also includes an unconventional nucleotide and an appropriate enzyme for degradation of nucleic acid polymers containing the unconventional nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
A method for determining the position of at least one selected species of nucleotide within a region of interest in a target nucleic acid polymer in a sample comprising the steps of combining the sample with a reaction mixture to synthesize chain-extension products indicative of the positions of the selected species of nucleotide within the region of interest and evaluating the products thus produced, wherein the reaction mixture which is combined with the sample comprises an unconventional nucleotide and a first enzyme said first enzyme being effective to degrade polynucleotides incorporating the unconventional nucleotide.
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