DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides
AssigneeShanghai Mendel DNA Center Co., Ltd.
InventorsGuoFan Hong, Wei-hua Huang
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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.
Claims26
A method of sequencing a DNA strand comprising the steps of:
- i) hybridizing a primer to a DNA template to be sequenced;
- ii) extending the primer using a modified DNA polymerase in the presence of adequate amounts of nucleotide bases dATP, dGTP, dCTP and dTTP, or their analogs, and fluorescent dye-labeled dideoxynucleotide terminators,
- wherein the modified DNA polymerase during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective discrimination against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state 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,
- under such conditions that the DNA strand is sequenced.
A method of dye-labeled primer sequencing of a DNA strand comprising the steps of:
- i) hybridizing a fluorescent dye-labeled primer to a DNA template to be sequenced;
- ii) extending the primer using a modified DNA polymerase in the presence of adequate amounts of nucleotide bases dATP, dGTP, dCTP and dTTP, or their analogs, and dideoxynucleotide terminators,
- wherein the modified DNA polymerase during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective discrimination against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state 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,
- under such conditions that the DNA strand is sequenced.
A method of ralioiostope-labeled sequencing a DNA strand comprising the steps of:
- i) providing four separate tubes, each containing DNA template to be sequenced, a primer, dATP, dTTP, dCTP and dGTP, wherein one of dATP, dTTP, dCTP or dGTP is labeled with a radioisotope, and a modified DNA polymerase which during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective disc nation against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state 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,
- wherein each tube contains an appropriate amount of one of dATP, dTTP, dCTP or dGTP;
- ii) allowing DNA sequencing of the DNA template to be carried out;
- and
- iii) determining the sequence of the DNA template.
Dependencies are inferred from the claim text.