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US 9,353,411 B2Granted
Nucleic acid sequencing technique using a pH-sensing agent
AssigneeParallel Synthesis Technologies
InventorRobert C. Haushlater
Life of this patent
Estimating the term…
Abstract
The invention relates to a composition, method and apparatus for determining the sequence of a nucleic acid strand utilizing a pH-sensing agent.
Claims19
A composition for sequencing nucleic acids comprising:
- (a) a solid surface comprising a reactive group;
- (b) a strand of nucleic acid;
- (c) a pH-sensing agent;
- (d) a nucleic acid polymerase;
- (e) at least one dNTP reagent;
- and
- (f) a reaction solution;
- wherein said pH-sensing agent comprises a pH-sensitive dye, the pH-sensitive dye being non-fluorescent at basic pH and fluorescent at neutral or acidic pH.
5 more elements
A composition for sequencing nucleic acids comprising:
- (a) a solid surface comprising a reactive group;
- (b) a strand of nucleic acid;
- (c) a pH-sensing agent;
- (d) a nucleic acid polymerase;
- (e) at least one dNTP reagent;
- and
- (f) a reaction solution;
- wherein said pH-sensing agent is bound to said surface via a bond selected from the group consisting of covalent bond, electrostatic bond, hydrogen bond or combinations thereof.
5 more elements
A composition for sequencing nucleic acids comprising:
- (a) a solid surface comprising a reactive group;
- (b) a strand of nucleic acid;
- (c) a pH-sensing dye;
- (d) a nucleic acid polymerase;
- (e) at least one dNTP reagent;
- and
- (f) a reaction solution;
- wherein said strand of nucleic acid comprises a nucleic acid bound to the solid surface and wherein said pH-sensing dye is adjacent to said nucleic acid bound to the solid surface.
5 more elements
A method for sequencing DNA comprising the steps of:
- (a) providing a pH-sensing agent and a reactive surface;
- (b) providing a nucleic acid priming sequence;
- (c) attaching said nucleic acid priming sequence to said reactive surface;
- (d) providing a nucleic acid to be sequenced;
- (e) providing a polymerase enzyme and a reaction solution;
- (f) adding said polymerase enzyme, said reaction solution, and said nucleic acid to be sequenced to the surface containing said pH-sensitive agent and said surface-bound primer;
- (g) resetting said pH-sensing agent by washing with a pH resetting solution, which resets the pH-sensing agent;
- (h) providing individual solutions of four dNTP reagents selected from the group consisting of:
- dATP, dGTP, dCTP, and dTTP;
- (i) adding said individual solutions of four dNTP reagents individually and sequentially washing with said pH resetting solution between each addition;
- (j) measuring a change in the properties of the pH-sensing agent after each addition of each dNTP;
- (k) correlating said change in the properties of the pH-sensing agent with the type and amount of each dNTP added;
- (l) using said change in properties to determine a nature and an amount of dNTP incorporated at each step;
- (m) determining the nucleotide sequence from the dNTP incorporation data;
- and
- (n) repeating steps (g) through (k) until the order of nucleotides in the nucleic acid strand is determined.
13 more elements
A composition for sequencing nucleic acids comprising:
- (a) a solid surface comprising a reactive group;
- (b) a strand of nucleic acid;
- (c) a pH-sensing agent;
- (d) a nucleic acid polymerase;
- (e) at least one dNTP reagent;
- and
- (f) a reaction solution;
- wherein said pH-sensing agent is selected from the group consisting of:
- Malachite green oxalate;
- Brilliant green;
- Eosin yellowish;
- Erythrosine B;
- Methyl green;
- Methyl violet;
- Picric acid;
- Cresol red;
- Crystal violet;
- m-Cresol purple;
- Thymol blue;
- p-Xylenol blue;
- Eosin, bluish;
- Quinaldine red;
- 2,4-Dinitro phenol;
- 4-(Dimethylamino) azo;
- Bromochlorophenol blue;
- Bromophenol blue;
- Congo red;
- Methyl orange;
- Bromocresol green;
- 2,5-Dinitrophenol;
- Alizarin sulphonic acid;
- Methyl red;
- Chlorophenol red;
- Litmus;
- Bromocresol purple;
- Bromophenol red;
- 4-Nitrophenol;
- Bromoxylenol blue;
- Bromothymol blue;
- Phenol red;
- 3-Nitrophenol;
- Neutral red;
- Creosol red;
- 1-Naphtholphthalein;
- m-Cresol purple;
- Thymol blue;
- p-Xylenol blue;
- Phenolphthalein;
- Thymolphthalein;
- Alkali blue;
- Alizarin yellow GG;
- Indigo carmine;
- Epsilon blue;
- and Titan yellow.
51 more elements
A method for sequencing DNA comprising the steps of:
- (a) providing a pH-sensing agent and a reactive surface;
- (b) providing a nucleic acid to be sequenced;
- (c) attaching said nucleic acid to said reactive surface;
- (d) providing a nucleic acid priming sequence;
- (e) providing a polymerase enzyme and a reaction solution;
- (f) adding said polymerase enzyme, said reaction solution, and said priming sequence to the surface containing said pH-sensitive agent and said surface-bound nucleic acid to be sequenced;
- (g) resetting said pH-sensing agent by washing with a pH resetting solution, which resets the pH-sensing agent;
- (h) providing individual solutions of four dNTP reagents selected from the group consisting of:
- dATP, dGTP, dCTP, and dTTP;
- (i) adding said individual solutions of four dNTP reagents individually and sequentially washing with said pH resetting solution between each addition;
- (j) measuring a change in the properties of the pH-sensing agent after each addition of each dNTP;
- (k) correlating said change in the properties of the pH-sensing agent with the type and amount of each dNTP added;
- (l) using said change in properties to determine a nature and an amount of dNTP incorporated at each step;
- (m) determining the nucleotide sequence from the dNTP incorporation data;
- and
- (n) repeating steps (g) through (k) until the order of nucleotides in the nucleic acid strand is determined.
13 more elements
Dependencies are inferred from the claim text.