Nucleic acid sequencing technique using a pH-sensing agent

• US 9,353,411 B2
• Filed: 03/30/2012
• Issued: 05/31/2016
• Est. Priority Date: 03/30/2011
• Status: Active Grant

• Current Assignee: Parallel Synthesis Technologies, Inc.
• Original Assignee: Parallel Synthesis Technologies
• Inventors: Haushlater, Robert C.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/436,465
• Publication Number:
  US 20130005583A1
• Time in Patent Office: 1,523 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

The invention relates to a composition, method and apparatus for determining the sequence of a nucleic acid strand utilizing a pH-sensing agent.

Claims

1. 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.

2. A composition of claim 1, wherein said solid surface is selected from the group consisting of agarose, silica, organic polymers, glass, inorganic materials, phosphors, clay and combinations thereof.

3. A composition of claim 1, wherein said reactive group is selected from the group consisting of C—

OH, —

CO₂H, —

NH₂, —

SH, —

CONH₂, —

CO₂CH₃, epoxides, alcohols, carboxylic acids, organic esters, amides, amines, alky or aryl halides, sulfates, phosphates, silanols, hydroxide or combinations thereof.

4. A composition of claim 3, wherein said reactive group is attached to a material selected from the group consisting of molecules, polymers, colloids, nanoparticles, and combinations thereof.

5. A composition of claim 1, wherein said strand of nucleic acid is bound to said surface via a bond selected from the group consisting of covalent bond, electrostatic bond, hydrogen bond or combinations thereof.

6. A composition of claim 1, wherein the components in said reaction solution comprises a PCR mix.

7. 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.

8. 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.

9. 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.

10. The method of claim 9, wherein said nucleic acid to be sequenced is DNA.

11. The method of claim 9, wherein said change in the properties of the pH-sensitive agent are compared to a second control sample that does not include said polymerase enzyme.

12. The method of claim 9, wherein the nucleic acid to be sequenced and pH-sensitive agent are attached to said surface by covalent, electrostatic or hydrogen bonding interactions.

13. The method of claim 9, wherein said nucleic acid primer is attached to the surface by hybridizing it to a previously immobilized complementary DNA strand previously bound to the surface.

14. The method of claim 9, wherein said nucleic acid to be sequenced is attached to the surface by hybridizing it to a complementary DNA strand previously bound to the surface.

15. The method of claim 9, wherein the nucleic acid to be sequenced is attached to said surface.

16. The method of claim 9, wherein the pH-sensing agent is covalently bound to said surface.

17. The method of claim 9, wherein the pH-sensing agent is in solution adjacent to the nucleic acid to being be sequenced.

18. 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.

19. 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.