Secondary Structure Defining Database And Methods For Determining Identity And Geographic Origin Of An Unknown Bioagent Thereby

• US 20110172925A1
• Filed: 10/31/2007
• Published: 07/14/2011
• Est. Priority Date: 06/26/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hofstadler, Steven A., Sampath, Rangarajan, Griffey, Richard H., McNeil, John, Ecker, David J., Crooke, Stanley T.
• Granted Patent: US 8,380,442 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/10 Nucleic acid folding
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: G16B 99/00 Subject matter not provided for in other groups of this subclass
• CPC Class Codes: G16H 50/80 for detecting, monitoring or modelling epidemics or pandemics, e.g. flu
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The present invention relates generally to the field of investigational bioinformatics and more particularly to secondary structure defining databases. The present invention further relates to methods for interrogating a database as a source of molecular masses of known bioagents for comparing against the molecular mass of an unknown or selected bioagent to determine either the identity of the selected bioagent, and/or to determine the origin of the selected bioagent. The identification of the bioagent is important for determining a proper course of treatment and/or irradication of the bioagent in such cases as biological warfare. Furthermore, the determination of the geographic origin of a selected bioagent will facilitate the identification of potential criminal identity.

Claims

1-20. -20. (canceled)

21. A method of identifying a bioagent, comprising:

a) determining a molecular mass of a nucleic acid molecule derived from a bioagent by mass spectrometry;

b) determining the base composition of said nucleic acid molecule from said molecular mass; and

c) identifying said bioagent by comparing said determined base composition to a database of base compositions from a plurality of different bioagents, wherein said plurality of different bioagents comprises bioagents from two or more different genera.

22. The method of claim 21, wherein said bioagent is selected from the group of bioagents consisting of a viral bioagent, a bacterial bioagent, a fungal bioagent, a protozoal bioagent, a parasitic bioagent, a mammalian bioagent, and a human bioagent.

23. The method of claim 21, wherein said bioagent is selected from the group of bioagents consisting of an evolving bioagent, a mutating bioagent, a recombinant bioagent, and an engineered bioagent.

24. The method of claim 21, wherein said bioagent is not previously known to exist.

25. The method of claim 21, wherein said bioagent is identified in a sample.

26. The method of claim 25, wherein said sample is selected from the group consisting of a body fluid sample, an environmental sample, a culture media sample, a manufacturing sample, and a forensic sample.

27. The method of claim 21, wherein said nucleic acid is selected from the group consisting of genomic DNA, RNA, DNA that is complementary to RNA, DNA that is synthesized from RNA, double-stranded DNA, single stranded DNA, DNA that is the product of amplification, DNA that is fragmented, nuclear DNA, mitochondrial DNA, cytoplasmic DNA and extracellular DNA.

28. The method of claim 21, wherein said bioagent is identified at the genus, species, sub-species, strain, sub-type or nucleotide polymorphism levels.

29. The method of claim 21, wherein said mass spectrometry is ESI mass spectrometry.

30. The method of claim 21, wherein said base composition comprises identification of the number of adenine (A), cytosine (C), thymine (T), guanine (G) and uracil (U) residues, analogs thereof and/or mass tag residues thereof in said nucleic acid molecule.

31. The method of claim 21, wherein said base composition is determined without sequencing said nucleic acid molecule.

32. The method of claim 21, wherein said comparing comprises identifying a match between said determined base composition and at least one entry within said database of base compositions from a plurality of different bioagents.

33. The method of claim 21, wherein said comparing step is performed by a computer.

34. The method of claim 33, wherein said computer identifies a match between said determined base composition of said nucleic acid molecule and one or more entries in database of base compositions from a plurality of different bioagents with a probability algorithm.

35. The method of claim 21, wherein said database of base compositions comprises at least 10 base compositions.

36. The method of claim 21, wherein said database of base compositions comprises at least 20 base compositions.

37. The method of claim 21, wherein said database of base compositions comprises at least 30 base compositions.

38. The method of claim 21, wherein said database of base compositions comprises at least 40 base compositions.

39. The method of claim 21, wherein said database of base compositions comprises at least 50 base compositions.

40. The method of claim 21, wherein said database of base compositions comprises at least 60 base compositions.

41. The method of claim 21, wherein said database of base compositions comprises at least 70 base compositions.

42. The method of claim 21, wherein said database of base compositions comprises at least 80 base compositions.

43. The method of claim 21, wherein said database of base compositions comprises at least 90 base compositions.

44. The method of claim 21, wherein said database of base compositions comprises at least 100 base compositions.

45. The method of claim 21, wherein said database of base compositions comprises at least 500 base compositions.

46. The method of claim 21, wherein said database of base compositions comprises at least 1000 base compositions.

47. The method of claim 21, wherein said nucleic acid molecule comprises a variable sequence region flanked by conserved sequence regions.

48. The method of claim 21, wherein said nucleic acid molecule is an amplification product.

49. The method of claim 48, wherein said amplification product is produced by polymerase chain reaction.

50. The method of claim 48, wherein said amplification product is produced by contacting a sample comprising said bioagent with two or more oligonucleotide primers that hybridize to sequence regions of said nucleic acid molecule that are conserved among different bioagents, wherein said conserved sequence regions flank a variable sequence region to produce an amplification product.

51. The method of claim 50, further comprising purifying molecules from said sample prior to producing said amplification product.

52. The method of claim 50, further comprising purifying said amplification product.

53. The method of claim 50, wherein said sample is contacted with two or more oligonucleotide primers configured to identify two or more bioagents of different genus.

54. The method of claim 50, wherein said sample is contacted with two or more oligonucleotide primers configured to identify two or more bioagents of different species.

55. The method of claim 50, further comprising modifying nucleic acid in said sample prior to producing said amplification product.

56. The method of claim 50, wherein said two or more oligonucleotide primers are about 15-35 nucleobases in length, and wherein said two or more oligonucleotide primers comprise at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identify with said conserved sequence regions.

57. The method of claim 50, wherein said amplification product is 45 to 200 nucleobases in length.

60. The method of claim 50, wherein at least one of said two or more oligonucleotide primers comprises at least one molecular mass modifying tag.

61. The method of claim 50, wherein at least one of said two or more oligonucleotide primers comprises at least one modified nucleobase.

62. The method of claim 61, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

63. The method of claim 61, wherein said modified nucleobase is a mass modified nucleobase.

64. The method of claim 63, wherein said mass modified nucleobase is 5-Iodo-Cytosine (5-Iodo-C).

65. The method of claim 61, wherein said modified nucleobase is a universal nucleobase.

66. The method of claim 65, wherein said universal nucleobase is inosine.

67. The method of claim 50, wherein said two or more oligonucleotide primers comprise multiple oligonucleotide primer sets configured for identification of diverse bioagents.

68. The method of claim 50, wherein said conserved sequence regions are within a gene.

69. The method of claim 50, wherein said conserved sequence regions are within a coding region of a gene.

70. The method of claim 50, wherein said conserved sequence regions are within a regulatory region of a gene.

71. The method of claim 50, wherein said variable sequence region is within a gene.

72. The method of claim 50, wherein said variable sequence region is within a coding region of a gene.

73. The method of claim 50, wherein said variable sequence region is within a regulatory region of a gene.

74. The method of claim 50, wherein said conserved sequence regions are about 10 to 100 nucleobases in length.

76. The method of claim 50, wherein said identifying said bioagent requires two or more oligonucleotide primer pairs.

77. The method of claim 50, wherein said database of base compositions from a plurality of different bioagents comprises base compositions of genus specific amplification products, family specific amplification products, species specific amplification products, strain specific amplification products, sub-type specific amplification products, or nucleotide polymorphism specific amplification products produced with said two or more oligonucleotide primers, wherein one or more matches between said determined base composition of said amplification product and one or more entries in said database identifies said bioagent, classifies a major classification of said bioagent, or differentiates between subgroups of known and unknown bioagents.

78. The method of claim 21, wherein said database of base compositions comprises base composition information for at least 3 different bioagents.

79. The method of claim 21, wherein said database of base compositions comprises base composition information for at least 4 different bioagents.

80. The method of claim 21, wherein said database of base compositions comprises base composition information for at least 8 different bioagents.

81. The method of claim 21, wherein said database of base compositions comprises base composition information for at least 19 different bioagents.

82. The method of claim 21, wherein said database of base compositions comprises base composition information for at least 30 different bioagents.

83. The method of claim 21, wherein said database of base compositions comprises at least 12 unique base compositions.

84. The method of claim 21, wherein said database of base compositions comprises at least 40 unique base compositions.

85. The method of claim 21, wherein said database of base compositions comprises base composition information for a bioagent from two or more genera selected from the group consisting of Acinetobacter, Aeromonas, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

86. The method of claim 21, wherein said database of base compositions comprises base composition information for a bioagent from each of the genera of Acinetobacter, Aeromonas, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, and Yersinia.

87. The method of claim 21, wherein said database of base compositions comprises base composition information for a bioagent from two or more orders or families selected from the group consisting of Smallpox virus, Arenavirus, Bunyaviruses, Mononegavirales, Picornaviruses, Astroviruses, Calciviruses, Nidovirales, Flaviviruses, and Togaviruses.

88. The method of claim 21, wherein said database of base compositions comprises base composition information for a bioagent from each of the orders or families of Smallpox virus, Arenavirus, Bunyaviruses, Mononegavirales, Picornaviruses, Astroviruses, Calciviruses, Nidovirales, Flaviviruses, and Togaviruses.

89. The method of claim 21, wherein said base compositions in said database are associated with bioagent identity.

90. The method of claim 21, wherein said base compositions in said database are associated with bioagent geographic origin.

93. The method of claim 21, wherein said database is stored on a computer.

94. The method of claim 93, wherein said computer is a local computer.

95. The method of claim 93, wherein said computer is a remote computer.

96. The method of claim 21, wherein said identifying said bioagent comprises interrogation of said database with two or more different base compositions associated with said bioagent.

58. (canceled)

59. (canceled)

75. (canceled)

91. (canceled)

92. (canceled)

97. A system, comprising:

a) a molecular mass determination component;

b) a database component comprising nucleic acid base compositions from a plurality of bioagents, wherein said plurality of bioagents comprises bioagents from two or more different genera; and

c) a processor configured to convert molecular mass data to nucleic acid base composition data, wherein said processor is further configured to compare said base composition data from a bioagent to said database component comprising nucleic acid base compositions from a plurality of bioagents to identify said bioagent.

98. The system of claim 97, further comprising a nucleic acid purification component.

99. The system of claim 98, wherein said nucleic acid purification component comprises one or more buffer manipulations, one or more salt manipulations, one or more thermal manipulations, one or more pH manipulations, one or more mechanical manipulations, one or more centrifugation manipulations, or one or more magnetic manipulations.

100. The system of claim 97, further comprising a nucleic acid amplification component.

101. The system of claim 100, wherein said nucleic acid amplification component comprises a thermocycler.

102. The system of claim 100, wherein said nucleic acid amplification component further comprises one or more salts, one or more buffers, one or more purified oligonucleotide primers, one or more deoxynucleotide triphosphates (dNTPS), or one or more enzymes.

103. The system of claim 97, wherein said molecular mass determination component comprises a mass spectrometer.

104. The system of claim 103, wherein said mass spectrometer is an ESI mass spectrometer.

105. The system of claim 97, wherein said base composition identification component comprises a processor.

106. The system of claim 105, further comprising a computer program on a computer readable medium configured to direct said processor to coordinate the operation of said nucleic acid component, said base composition determination component, and said base composition identification component.

107. The system of claim 105, wherein said processor is configured to process mass spectrometry data to base composition data.

108. The system of claim 105, wherein said processor is configured to process base composition data to identify a bioagent.

109. The system of claim 97, wherein said database of base compositions comprises at least 10 base compositions.

110. The system of claim 97, wherein said database of base compositions comprises at least 20 base compositions.

111. The system of claim 97, wherein said database of base compositions comprises at least 30 base compositions.

112. The system of claim 97, wherein said database of base compositions comprises at least 40 base compositions.

113. The system of claim 97, wherein said database of base compositions comprises at least 50 base compositions.

114. The system of claim 97, wherein said database of base compositions comprises at least 60 base compositions.

115. The system of claim 97, wherein said database of base compositions comprises at least 70 base compositions.

116. The system of claim 97, wherein said database of base compositions comprises at least 80 base compositions.

117. The system of claim 97, wherein said database of base compositions comprises at least 90 base compositions.

118. The system of claim 97, wherein said database of base compositions comprises at least 100 base compositions.

119. The system of claim 97, wherein said database of base compositions comprises at least 500 base compositions.

120. The system of claim 97, wherein said database of base compositions comprises at least 1000 base compositions.

121. The system of claim 97, wherein said plurality of bioagents comprises bioagents that differ by genus, species, sub-species, strain, sub-type or nucleotide polymorphism.

122. The system of claim 97, wherein said plurality of bioagents comprises one or more viral bioagents, one or more bacterial bioagents, one or more fungal bioagents, one or more protozoal bioagents, one or more parasitic bioagents, one or more mammalian bioagents, or one or more human bioagents.