Novel sulfurylase-luciferase fusion proteins and thermostable sulfurylase

• US 20050124022A1
• Filed: 10/30/2002
• Published: 06/09/2005
• Est. Priority Date: 10/30/2001
• Status: Abandoned Application

• Current Assignee: Maithreyan Srinivasan, Michael Reifler
• Original Assignee: Maithreyan Srinivasan, Michael Reifler
• Inventors: Srinivasan, Maithreyan, Reifler, Michael
• Application Number:
  US10/494,073
• Publication Number:
  US 20050124022A1
• Time in Patent Office: Days
• US Class Current: 435/21
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)

Abstract

The present invention relates to the field of DNA recombinant technology. More specifically, this invention relates to fusion proteins comprising an ATP generating polypeptide joined to a polypeptide that converts ATP into a detectable entity. Accordingly, this invention focuses on sulfurylase-luciferase fusion proteins. This invention also relates to pharmaceutical compositions containing the fusion proteins and methods for using them.

Claims

1. A fusion protein comprising an ATP generating polypeptide bound to a polypeptide which converts ATP to an entity that is detectable.

2. The fusion protein of claim 1 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

3. The fusion protein of claim 2 wherein the ATP sulfurylase is a thermostable sulfurylase comprising the nucleotide sequence of SEQ ID NO:

1.

4. The fusion protein of claim 3 wherein the nucleotide sequence encodes the polypeptide sequence of SEQ ID NO:

2.

5. The fusion protein of claim 3 wherein the thermostable sulfurylase is active at room temperature.

6. The fusion protein of claim 2 wherein the ATP sulfurylase is from a thermophile.

7. The fusion protein of claim 6 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

8. The fusion protein of claim 1 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

9. The fusion protein of claim 8 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

10. The fusion protein of claim 9 wherein the animal is selected from the group consisting of mammal, rodent, insect, worm, mollusk, reptile, bird and amphibian.

11. The fusion protein of claim 9 wherein the plant is selected from the group consisting of Arabidopsis thaliana, Brassica napus, Allium sativum, Amaranthus caudatus, Hevea brasiliensis, Hordeum vulgare, Lycopersicon esculentum, Nicotiana tabacum, Oryza sativum, Pisum sativum, Populus trichocarpa, Solanum tuberosum, Secale cereale, Sambucus nigra, Ulmus americana or Triticum aestivum.

12. The fusion protein of claim 9 wherein the fungus is Penicillum chrysogenum, Stachybotrys chartarum, Aspergillus fumigatus, Podospora anserina, Trichoderma reesei and Riftia pachyptila.

13. The fusion protein of claim 9 wherein the yeast is Saccharomyces cerevisiae, Candida tropicalis, Candida lypolitica, Candida utilis, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida spp., Pichia spp. and Hansenula spp..

14. The fusion protein of claim 8 wherein the prokaryote is bacteria or archaea.

15. The fusion protein of claim 14 wherein the bacteria selected from the group consisting of E. coli, B. subtilis, Streptococcus gordonii, flavobacteria and green sulfur bacteria.

16. The fusion protein of claim 14 wherein the archaea is selected from the group consisting of Sulfolobus, Thermococcus, Methanobacterium, Halococcus, Halobacterium and Methanococcus jannaschii.

17. The fusion protein of claim 1 wherein the detectable entity is selected from the group consisting of chemiluminescence, bioluminescence and fluorescence.

18. The fusion protein of claim 1 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

19. The fusion protein of claim 18 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

20. The fusion protein of claim 1 which further comprises an affinity tag.

21. The fusion protein of claim 20 wherein the affinity tag is selected from the group consisting of N-terminal poly-histidine, BCCP, protein A, glutathione S transferase, substance P and streptavidin binding peptide.

22. The fusion protein of claim 1 wherein the polypeptides are joined by a linker.

23. The fusion protein of claim 22 wherein the linker is an ala-ala-ala linker.

24. The fusion protein of claim 1 wherein the ATP generating polypeptide is N-terminal to the ATP converting polypeptide.

25. The fusion protein of claim 1 wherein the ATP converting polypeptide is N-terminal to the ATP generating polypeptide.

51. The fusion protein of claim 1 bound to a mobile support.

52. The fusion protein of claim 51 wherein the fusion protein is attached by a covalent or non-covalent interaction.

53. The fusion protein of claim 52 wherein the fusion protein is attached by a linkage selected from the group consisting of a metal, a CO²⁺-hexahistidine complex, a Ni²⁺-hexahistidine complex, a biotin binding protein, a glutathione S-transferase/glutathione complex, a monoclonal antibody/antigen complex, a maltose binding protein/maltose complex and pluronic coupling.

54. The fusion protein of claim 53 wherein the biotin binding protein is selected from the group consisting of NEUTRAVIDIN™

modified avidin, streptavidin and avidin.

55. The fusion protein of claim 51 wherein the mobile support is selected from the group consisting of a bead, optical fiber and glass surface.

56. The fusion protein of claim 55 wherein the bead is a nickel-agarose bead or a MPG-Streptavidin bead.

57. The fusion protein of claim 51 wherein the fusion protein is bound to the mobile support in a 1:

3 ratio of protein to mobile support.

58. The fusion protein of claim 51 wherein the fusion protein is a sulfurylase-luciferase fusion protein.

26. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1, 3 and 5.

27. An isolated polypeptide comprising an amino acid sequence selected from the group consising of SEQ ID NOs:

2, 4 and 6.

28. A fusion protein comprising a sulfurylase polypeptide bound to a luciferase polypeptide and at least one affinity tag.

29. The fusion protein of claim 28 wherein the fusion protein comprises the sequence of SEQ ID NO:

4.

30. The fusion protein of claim 28 wherein the fusion protein is encoded by a nucleic acid comprising the sequence of SEQ ID NO:

3.

31. A fusion protein comprising a thermostable sulfurylase bound to at least one affinity tag.

32. The fusion protein of claim 31 wherein the fusion protein comprises the sequence of SEQ ID NO:

6.

33. The fusion protein of claim 31 wherein the fusion protein is encoded by a nucleic acid comprising the sequence of SEQ ID NO:

5.

34. A recombinant polynucleotide that comprises a coding sequence for a fusion protein having an ATP generating polypeptide sequence and an ATP converting polypeptide sequence.

35. The recombinant polynucleotide sequence of claim 34 wherein the ATP generating polypeptide is ATP sulfurylase.

36. The recombinant polynucleotide sequence of claim 34 wherein the ATP converting polypeptide is luciferase.

37. The recombinant polynucleotide of claim 34 wherein the ATP generating polypeptide is N-terminal to the ATP converting polypeptide.

38. The recombinant polynucleotide of claim 34 wherein the ATP converting polypeptide is N-terminal to the ATP generating polypeptide.

39. An expression vector for expressing a fusion protein, said vector comprising a coding sequence for a fusion protein having:

(i) a regulatory sequence, (ii) a first polypeptide sequence of an ATP generating polypeptide and (iii) a second polypeptide sequence that converts ATP to an entity which is detectable.

40. The expression vector of claim 39 wherein the vector further comprises an affinity tag.

41. The expression vector of claim 39 wherein the ATP generating polypeptide is ATP sulfurylase.

42. The expression vector of claim 39 wherein the ATP converting polypeptide is luciferase.

43. The expression vector of claim 39 wherein the regulatory element is an enhancer or a promoter.

44. The expression vector of claim 43 wherein the promoter is a constitutive promoter or an inducible promoter.

45. A transformed host cell which contains the expression vector of claim 39.

46. The transformed host cell of claim 45 wherein the host cell is a eukaryotic cell.

47. The transformed host cell of claim 46 wherein the eukaryotic cell is human, rat or mouse.

48. The transformed host cell of claim 45 wherein the host cell is a prokaryotic cell.

49. The transformed host cell of claim 48 wherein the prokaryotic cell is bacteria.

50. A purified fusion protein expressed by cells transformed with an expression vector of claim 39.

189. A kit comprising a sulfurylase-luciferase fusion protein expression vector as claimed in claim 39.

59. 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 PPi with an ATP generating polypeptide-ATP converting polypeptide fusion protein 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.

60. The method of claim 59 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

61. The method of claim 60 wherein the ATP sulfurylase is a thermostable sulfurylase.

62. The method of claim 60 wherein the ATP sulfurylase is from a thermophile.

63. The method of claim 62 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

64. The method of claim 59 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

65. The method of claim 64 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

66. The method of claim 59 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

67. The method of claim 66 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

68. The method of claim 59 which further comprises an affinity tag.

69. A method according to claim 59, wherein the amount of inorganic pyrophosphate is measured by

(a) adding adenosine-5′

-phosphosulfate to the feedstock;

(b) combining the recovered feedstock containing adenosine-5′

-phosphosulfate with an ATP generating polypeptide-ATP converting polypeptide fusion protein such that any inorganic pyrophosphate in the recovered feedstock and the adenosine-5′

-phosphosulfate will react to the form ATP and sulfate;

(c) combining the ATP, sulfate, and said fusion protein-containing feedstock with luciferin in the presence of oxygen such that the ATP is consumed to produced AMP, inorganic pyrophosphate, carbon dioxide and light; and

(d) measuring the amount of light produced.

70. The method of claim 69 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

71. The method of claim 70 wherein the ATP sulfurylase is a thermostable sulfurylase.

72. The method of claim 70 wherein the ATP sulfurylase is from a thermophile.

73. The method of claim 72 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

74. The method of claim 69 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

75. The method of claim 74 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

76. The method of claim 69 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

77. The method of claim 76 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

78. The method of claim 69 which further comprises an affinity tag.

79. The method according to claim 59 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 is determined by reacting the inorganic pyrophosphate-containing feedstock with an ATP generating polypeptide-ATP converting polypeptide fusion protein thereby producing light in an amount proportional to the amount of inorganic pyrophosphate, and measuring the amount of light produced.

80. The method of claim 79 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

81. The method of claim 80 wherein the ATP sulfurylase is a thermostable sulfurylase.

82. The method of claim 80 wherein the ATP sulfurylase is from a thermophile.

83. The method of claim 82 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

84. The method of claim 79 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

85. The method of claim 84 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

86. The method of claim 79 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

87. The method of claim 86 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

88. The method of claim 79 which further comprises an affinity tag.

89. A method for sequencing a nucleic acid, the method comprising:

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded circular nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(c) annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′

end of said sequencing primer, a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct with the use of an ATP generating polypeptide-ATP converting polypeptide fusion protein, thereby determining the sequence of the nucleic acid.

90. The method of claim 89 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

91. The method of claim 90 wherein the ATP sulfurylase is a thermostable sulfurylase.

92. The method of claim 90 wherein the ATP sulfurylase is from a thermophile.

93. The method of claim 92 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

94. The method of claim 89 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

95. The method of claim 94 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

96. The method of claim 89 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

97. The method of claim 96 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

98. The method of claim 89 which further comprises an affinity tag.

99. A method for sequencing a nucleic acid, the method comprising:

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) annealing a first amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing a second amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, when the predetermined nucleotide triphosphate is incorporated onto the 3′

end of the sequencing primer, to yield a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct with the use of an ATP generating polypeptide-ATP converting polypeptide fusion protein, thereby determining the sequence of the nucleic acid at each reaction site that contains a nucleic acid template.

100. The method of claim 99 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

101. The method of claim 100 wherein the ATP sulfurylase is a thermostable sulfurylase.

102. The method of claim 100 wherein the ATP sulfurylase is from a thermophile.

103. The method of claim 102 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

104. The method of claim 99 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

105. The method of claim 104 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

106. The method of claim 99 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

107. The method of claim 106 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

108. The method of claim 99 which further comprises an affinity tag.

109. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) adding an activated nucleotide 5′

-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′

-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′

-triphosphate precursor onto the 3′

-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′

-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

(c) detecting whether or not the nucleoside 5′

-triphosphate precursor was incorporated into the primer strands through detection of a sequencing byproduct with an ATP generating polypeptide-ATP converting polypeptide fusion protein, thus indicating that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′

-triphosphate precursor; and

(d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′

-triphosphate precursor of known nitrogenous base composition; and

(e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.

110. The method of claim 109 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

111. The method of claim 110 wherein the ATP sulfurylase is a thermostable sulfurylase.

112. The method of claim 110 wherein the ATP sulfurylase is from a thermophile.

113. The method of claim 112 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

114. The method of claim 109 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

115. The method of claim 114 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

116. The method of claim 109 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

117. The method of claim 116 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

118. The method of claim 109 which further comprises an affinity tag.

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

(a) introducing a plurality of template nucleic acid polymers into a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, each reaction chamber having 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) detecting the formation of inorganic pyrophosphate with an ATP generating polypeptide-ATP converting polypeptide fusion protein to determine the identity of each nucleotide in the complementary polymer and thus the sequence of the template polymer.

120. The method of claim 119 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

121. The method of claim 120 wherein the ATP sulfurylase is a thermostable sulfurylase.

122. The method of claim 120 wherein the ATP sulfurylase is from a thermophile.

123. The method of claim 122 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

124. The method of claim 119 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

125. The method of claim 124 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

126. The method of claim 119 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

127. The method of claim 126 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

128. The method of claim 119 which further comprises an affinity tag.

129. A method of identifying the base in a target position in a DNA sequence of sample DNA, wherein:

(a) sample DNA is disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers,

(b) an extension primer is provided which hybridizes to said immobilized single-stranded DNA at a position immediately adjacent to said target position;

(c) said immobilized single-stranded DNA is subjected to a polymerase reaction in the presence of a predetermined nucleotide triphosphate, wherein if the predetermined nucleotide triphosphate is incorporated onto the 3′

end of said sequencing primer then a sequencing reaction byproduct is formed; and

(d) identifying the sequencing reaction byproduct with an ATP generating polypeptide-ATP converting polypeptide fusion protein, thereby determining the nucleotide complementary to the base at said target position.

130. The method of claim 129 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

131. The method of claim 130 wherein the ATP sulfurylase is a thermostable sulfurylase.

132. The method of claim 130 wherein the ATP sulfurylase is from a thermophile.

133. The method of claim 132 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

134. The method of claim 129 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

135. The method of claim 134 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

136. The method of claim 129 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

137. The method of claim 136 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

138. The method of claim 129 which further comprises an affinity tag.

139. A method of identifying a base at a target position in a sample DNA sequence comprising:

(a) providing sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers;

(b) providing an extension primer which hybridizes to the sample DNA immediately adjacent to the target position;

(c) subjecting the sample DNA sequence and the extension primer to a polymerase reaction in the presence of a nucleotide triphosphate whereby the nucleotide triphosphate will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said nucleotide triphosphate being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture; and

(d) detecting the release of PPi with an ATP generating polypeptide-ATP converting polypeptide fusion protein to indicate which nucleotide is incorporated.

140. The method of claim 139 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

141. The method of claim 140 wherein the ATP sulfurylase is a thermostable sulfurylase.

142. The method of claim 140 wherein the ATP sulfurylase is from a thermophile.

143. The method of claim 142 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

144. The method of claim 139 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

145. The method of claim 144 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

146. The method of claim 139 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

147. The method of claim 146 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

148. The method of claim 139 which further comprises an affinity tag.

149. A method of identifying a base at a target position in a single-stranded sample DNA sequence, the method comprising:

(a) providing an extension primer which hybridizes to sample DNA immediately adjacent to the target position, said sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 um, said DNA being rendered single stranded either before or after being disposed in the reaction chambers;

(b) subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said predetermined deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture, (c) detecting any release of PPi with an ATP generating polypeptide-ATP converting polypeptide fusion protein to indicate which deoxynucleotide or dideoxynucleotide is incorporated;

characterized in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi—

detection enzyme.

150. The method of claim 149 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

151. The method of claim 150 wherein the ATP sulfurylase is a thermostable sulfurylase.

152. The method of claim 150 wherein the ATP sulfurylase is from a thermophile.

153. The method of claim 152 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

154. The fusion protein of claim 149 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

155. The method of claim 154 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

156. The method of claim 149 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

157. The method of claim 156 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

158. The method of claim 149 which further comprises an affinity tag.

159. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) converting PPi into light with a an ATP generating polypeptide-ATP converting polypeptide fusion protein;

(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(f) recording the variations of said electrical signals with time.

160. The method of claim 159 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

161. The method of claim 160 wherein the ATP sulfurylase is a thermostable sulfurylase.

162. The method of claim 160 wherein the ATP sulfurylase is from a thermophile.

163. The method of claim 162 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

164. The method of claim 159 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

165. The method of claim 164 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

166. The method of claim 159 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

167. The method of claim 166 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

168. The method of claim 159 which further comprises an affinity tag.

169. Method for sequencing a nucleic acid, the method comprising:

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded circular nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(c) converting PPi into a detectable entity with the use of an ATP generating polypeptide-ATP converting polypeptide fusion protein;

(d) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(e) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(f) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(g) recording the variations of said electrical signals with time.

170. The method of claim 169 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

171. The method of claim 170 wherein the ATP sulfurylase is a thermostable sulfurylase.

172. The method of claim 170 wherein the ATP sulfurylase is from a thermophile.

173. The method of claim 172 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

174. The method of claim 169 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

175. The method of claim 174 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

176. The method of claim 169 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

177. The method of claim 176 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

178. The method of claim 169 which further comprises an affinity tag.

179. A method for sequencing a nucleic acid, the method comprising:

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) converting PPi into a detectable entity with an ATP generating polypeptide-ATP converting polypeptide fusion protein;

(d) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(e) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(f) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(g) recording the variations of said electrical signals with time.

180. The method of claim 179 wherein the ATP generating polypeptide is selected from the group consisting of ATP sulfurylase, hydrolase and ATP synthase.

181. The method of claim 180 wherein the ATP sulfurylase is a thermostable sulfurylase.

182. The method of claim 180 wherein the ATP sulfurylase is from a thermophile.

183. The method of claim 182 wherein the thermophile is a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

184. The method of claim 179 wherein the ATP generating polypeptide and ATP converting polypeptide are from a eukaryote or a prokaryote.

185. The method of claim 184 wherein the eukaryote is selected from the group consisting of animal, plant, fungus and yeast.

186. The method of claim 179 wherein the ATP converting polypeptide is selected from the group consisting of luciferase, ecto-nucleoside diphosphate kinase and ATPase.

187. The method of claim 186 wherein the luciferase is selected from the group consisting of Photinus pyralis, Pyroplorus plagiophihalamus (Coleoptera), Luciola cruciata and Luciola lateralis.

188. The method of claim 179 which further comprises an affinity tag.

190. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence of SEQ ID NO;

2;

(b) a variant of a mature form of an amino acid sequence of SEQ ID NO;

2;

(c) an amino acid sequence of SEQ ID NO;

2;

(d) a variant of an amino acid sequence of SEQ ID NO;

2, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 4% of amino acid residues from said amino acid sequence; and

(e) an amino acid sequence of (a), (b), (c) or (d) further containing one or more conservative amino acid substitutions.

191. The polypeptide of claim 190 wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence of SEQ ID NO:

2.

192. The polypeptide of claim 190 wherein the amino acid sequence of said variant comprises one or more conservative amino acid substitution.

200. An antibody that binds immunospecifically to the polypeptide of claim 190.

193. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence of SEQ ID NO;

2;

(b) a variant of a mature form of an amino acid sequence of SEQ ID NO;

2, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 4% of the amino acid residues from the amino acid sequence of said mature form;

(c) an amino acid sequence of SEQ ID NO;

2;

(d) a variant of an amino acid sequence of SEQ ID NO;

2, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 4% of amino acid residues from said amino acid sequence;

(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence of SEQ ID NO;

2, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 4% of amino acid residues from said amino acid sequence; and

(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).

194. The nucleic acid molecule of claim 193 wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.

195. The nucleic acid molecule of claim 193 wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.

196. The nucleic acid molecule of claim 193 wherein the nucleic acid molecule comprises nucleotide sequence selected from the group consisting of:

(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 11% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;

(b) an isolated second polynucleotide that is a complement of the first polynucleotide; and

(c) a nucleic acid fragment of (a) or (b).

197. A vector comprising the nucleic acid molecule of claim 196.

198. The vector of claim 197, further comprising a promoter operably-linked to said nucleic acid molecule.

199. A cell comprising the vector of claim 197.

201. 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 PPi with a thermostable sulfurylase and a luciferase 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.

202. The method of claim 201 wherein said thermostable sulfurylase comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence of SEQ ID NO:

2.

203. The method of claim 201 wherein the thermostable sulfurylase is derived from a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

204. The method of claim 201 wherein the thermostable sulfurylase and the luciferase are joined in a fusion protein.

205. The method of claim 201 wherein the thermostable sulfurylase is joined to an affinity tag.

206. A method for sequencing a nucleic acid, the method comprising:

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded circular nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200;

(c) annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′

end of said sequencing primer, a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct with the use of a thermostable sulfurylase and a luciferase, thereby determining the sequence of the nucleic acid.

207. The method of claim 206 wherein said thermostable sulfurylase comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence of SEQ ID NO:

2.

208. The method of claim 206 wherein the thermostable sulfurylase is derived from a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

209. The method of claim 206 wherein the thermostable sulfurylase and the luciferase are joined in a fusion protein.

210. The method of claim 206 wherein the thermostable sulfurylase is joined to an affinity tag.

211. A method for sequencing a nucleic acid, the method comprising:

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) annealing a first amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing a second amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, when the predetermined nucleotide triphosphate is incorporated onto the 3′

end of the sequencing primer, to yield a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct with the use of a thermostable sulfurylase and a luciferase, thereby determining the sequence of the nucleic acid at each reaction site that contains a nucleic acid template.

212. The method of claim 211 wherein said thermostable sulfurylase comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence of SEQ ID NO:

2.

213. The method of claim 211 wherein the thermostable sulfurylase is derived from a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

214. The method of claim 211 wherein the thermostable sulfurylase and the luciferase are joined in a fusion protein.

215. The method of claim 211 wherein the thermostable sulfurylase is joined to an affinity tag.

216. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) adding an activated nucleotide 5′

-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′

-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′

-triphosphate precursor onto the 3′

-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′

-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

(c) detecting whether or not the nucleoside 5′

-triphosphate precursor was incorporated into the primer strands through detection of a sequencing byproduct with a thermostable sulfurylase and luciferase, thus indicating that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′

-triphosphate precursor; and

(d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′

-triphosphate precursor of known nitrogenous base composition; and

(e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.

217. The method of claim 216 wherein said thermostable sulfurylase comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence of SEQ ID NO:

2.

218. The method of claim 216 wherein the thermostable sulfurylase is derived from a thermophilic bacteria selected from the group consisting of Bacillus stearothermophilus, Thermus thermophilus, Bacillus caldolyticus, Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus, Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus, Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus and Thermomonospora fusca.

219. The method of claim 216 wherein the thermostable sulfurylase and the luciferase are joined in a fusion protein.

220. The method of claim 216 wherein the thermostable sulfurylase is joined to an affinity tag.