Pyrophosphate-based method and apparatus for sequencing nucleic acids

• US 4,971,903 A
• Filed: 03/25/1988
• Issued: 11/20/1990
• Est. Priority Date: 03/25/1988
• Status: Expired due to Fees

• Current Assignee: Edward Hyman
• Original Assignee: Edward Hyman
• Inventors: Hyman, Edward
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Chambers, Scott
• Application Number:
  US07/173,433
• Time in Patent Office: 970 Days
• Field of Search: 435/6, 435/91
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A method for sequencing nucleic acid polymers is provided in which the polymer to be sequenced acts as a template for the production of a complementary polymer by a polymerase enzyme. The template polymer is introduced into a polymerization environment in which production of the complementary polymer will occur if appropriate nucleotides are provided. The nucleotides are then provided to the polymerization environment one at a time in individual feedstocks. If the nucleotide in a feedstock is complementary to the next base in the template polymer, i.e., the unpaired base closest to the growing end of the complementary polymer, polymerization will occur lengthening the complementary polymer and releasing PPi. By separately recovering each feedstock and analyzing it for the presence of PPi, the sequence of the complementary polymer and thus the template polymer is determined.

Claims

1. A method for determining the nucleic acid sequence in a template nucleic acid polymer, comprising

(a) introducing the template nucleic acid polymer into a polymerization environment in which the nucleic acid polymer will act as a template polymer for the synthesis of a complementary nucleic acid polymer when nucleotides are added;

(b) successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released;

(c) separately recovering each of the feedstocks from the polymerization environment; and

(d) measuring the amount of inorganic pyrophosphate in each of the recovered feedstocks to determine the identity of each nucleotide in the complementary polymer and thus the sequence of the template polymer.

2. A method according to claim 1, wherein the amount of inorganic pyrophosphate is measured by

adding adenosine-5'"'"'-phosphosulfate to the feedstock;

combining the recovered feedstock containing the adenosine-5'"'"'-phosphosulfate with the enzyme ATP-sulfurylase such that any inorganic pyrophosphate in the recovered feedstock and the adenosine-5'"'"'-phosphosulfate will react to the form ATP and sulfate;

combining the ATP and sulfate-containing feedstock with luciferin and an ATP-dependent luciferase in the presence of oxygen such that the ATP is consumed to produce AMP, inorganic pyrophosphate, carbon dioxide and light; and

measuring the amount of light produced.

3. A method according to claim 1, wherein the template polymer is immobilized on a solid support.

4. A method according to claim 3, wherein the immobilized template polymer is complexed with a primer sequence.

5. A method according to claim 4, wherein the template-primer complex is bound to a nucleic acid polymerase.

6. A method according to claim 5, wherein the amount of inorganic pyrophosphate is measured by

adding adenosine-5'"'"'-phosphosulfate to the feedstock;

combining the recovered feedstock containing the adenosine-5'"'"'-phosphosulfate with the enzyme ATP sulfurylase such that any inorganic pyrophosphate in the recovered feedstock and the adenosine-5'"'"'-phosphosulfate will react to form ATP and sulfate;

combining the ATP and sulfate containing feedstock with luciferin and an ATP-dependent luciferase in the presence of oxygen such that the ATP is consumed to produce AMP, inorganic pyrophosphate, carbon dioxide and light; and

measuring the amount of light produced.

7. A method according to claim 1, wherein each feedstock comprises adenosine-5'"'"'-phosphosulfate and luciferin in addition to the selected nucleotide base, and the amount of inorganic pyrophosphate is determined by reacting the inorganic pyrophosphate-containing feedstock with ATP sulfurylase and then with an ATP-dependent luciferase thereby producing light in an amount proportional to the amount of inorganic pyrophosphate, and measuring the amount of light produced.

8. A method according to claim 7, further comprising the step of treating the feedstock to remove inorganic pyrophosphate prior to providing the feedstock to the polymerization environment.

9. A method according to claim 8, wherein the feedstock is treated with immobilized pyrophosphatase enzyme to remove inorganic pyrophosphate prior to providing the feedstock to the polymerization environment.

10. A method according to claim 7, further comprising the step of treating the feedstock to remove ATP prior to reacting the feedstock with the ATP sulfurylase.

11. A method according to claim 10, wherein the feedstock further comprises glycerol and wherein the feedstock is treated with glycerokinase to remove ATP.

12. A method according to claim 10, wherein the feedstock further comprises glucose and wherein the feedstock is treated with hexokinase to remove ATP.

13. A method according to claim 11, wherein the feedstock further comprises glucose and wherein the feedstock is treated with hexakinase to remove ATP.