Nucleic acid arrays to monitor water and other ecosystems

• US 9,126,165 B1
• Filed: 03/09/2007
• Issued: 09/08/2015
• Est. Priority Date: 04/24/2002
• Status: Expired due to Fees

• Current Assignee: University of North Carolina At Greensboro (University of North Carolina System)
• Original Assignee: University of North Carolina At Greensboro (University of North Carolina System)
• Inventors: Rublee, Parke A., Henrich, Vincent C. III
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US11/716,450
• Time in Patent Office: 3,105 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01J 8/00 Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

Disclosed are arrays for monitoring ecosystems, such as bodies of water, and methods and systems for making such arrays. In one embodiment, the array may include a plurality of oligonucleotides immobilized at known locations on a substrate, such that each location on the array is an oligonucleotide having a sequence derived from a single, predetermined operational taxonomic unit (OTU). The sequences immobilized on the array may be from known, or unknown organisms. Also disclosed are methods for identifying and isolating bioindicators diagnostic of specific ecosystems. The arrays and bioindicators of the invention may be used for rapid, and continual monitoring of ecosystems.

Claims

1. A device comprising an array, wherein the array comprises a plurality of oligonucleotides covalently immobilized at known locations of the array, and wherein each location on the array comprises an oligonucleotide having a sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 and SEQ ID NO;

106 or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 and SEQ ID NO;

106.

2. The device of claim 1, wherein the oligonucleotides at each location of the array range from about 30 to 200 nucleotides in length.

3. The device of claim 1, wherein the oligonucleotides at each location of the array range from about 40 to 100 nucleotides in length.

4. The device of claim 1, wherein the oligonucleotides at each location of the array are each about 50 nucleotides in length.

5. The device of claim 1, wherein at least one of the oligonucleotides comprises a eukaryotic ribosomal DNA sequence.

6. The device of claim 1, wherein at least one of the oligonucleotides comprises a prokaryotic ribosomal DNA sequence.

7. The device of claim 1, wherein at least one of the oligonucleotides further comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

25;

or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99, or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

25, or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99;

or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113.

8. The device of claim 1, wherein there are at least 10 different oligonucleotides, each at a distinct location on the array, and each of the at least 10 different oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

113.

9. The device of claim 1, wherein there are at least 50 different oligonucleotides, each at a distinct location on the array, and each of the at least 50 different oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

113.

10. The device of claim 1, wherein the individual oligonucleotides on the array range from 1 femtogram (fg) to about 10 micrograms (μ

g) at each location of the array.

28. The device of claim 1, wherein the plurality of oligonucleotides are derived from at least one of a prokaryotic ribosomal DNA sequence that corresponds to nucleotides 160-250 of the prokaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO:

320 or nucleotides 430-510 of a prokaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

321, or from at least one of a eukaryotic ribosomal DNA sequences that correspond to nucleotides 600 to 800 of a eukaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO;

325;

or nucleotides 1000 to 1100 of the eukaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

327.

30. The device of claim 1, wherein each oligonucleotide has a sequence that is specific to a single, predetermined microbial operational taxonomic unit, wherein an operational taxonomic unit is comprised of sequences having at least 97.5% identity to each other.

31. The device of claim 1, wherein each of the plurality of oligonucleotides is selected from at least one of a prokaryotic small subunit ribosomal DNA based on alignments with E. Coli V1, V2, V3 or V4 variable regions for each prokaryotic operational taxonomic unit, or at least one of a eukaryotic small subunit ribosomal DNA based on alignments with S. cerevisiae V1, V2, V3 or V4 variable regions for each eukaryotic operational taxonomic unit.

11. A system for monitoring an ecosystem comprising:

(a) an array comprising a plurality oligonucleotides covalently immobilized at known locations of the array, such that each location on the array comprises an oligonucleotide having a sequence as set forth in any one of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106; and

(b) polymerase chain reaction primers for amplifying DNA isolated from an ecosystem to generate oligonucleotides complementary to at least some of the plurality of oligonucleotides of the array to generate an amplified nucleic acid sample from the ecosystem.

12. The system of claim 11, wherein at least one of the oligonucleotides further comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

25, or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99, or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

25, or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99, or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113.

13. The system of claim 11, wherein at least two of the oligonucleotides comprises the sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

14. The system of claim 11, wherein at least four of the oligonucleotides comprises the sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

15. The system of claim 11, wherein at least one of the PCR primers comprises the sequence as set forth in any one of SEQ ID NO:

156-SEQ ID NO;

157, SEQ ID NO;

168-SEQ ID NO;

169, SEQ ID NO;

180-SEQ ID NO;

181, SEQ ID NO;

283-SEQ ID NO;

284, SEQ ID NO;

287-SEQ ID NO;

288, or SEQ ID NO;

300-SEQ ID NO;

301.

16. The system of claim 11, wherein at least one of the oligonucleotides comprises a eukaryotic ribosomal DNA sequence and at least one of the immobilized oligonucleotides comprises a prokaryotic ribosomal DNA sequence.

17. The system of claim 11, wherein each of the plurality of oligonucleotides is selected from at least one of a prokaryotic small subunit ribosomal DNA based on alignments with E. Coli V1, V2, V3 or V4 variable region for each prokaryotic operational taxonomic unit, or at least one of a eukaryotic small subunit ribosomal DNA based on alignments with S. cerevisiae V1, V2, V3 or V4 variable region for each eukaryotic operational taxonomic unit.

18. The system of claim 11, wherein there are at least 10 different oligonucleotides, each at a distinct location on the array, and each of the at least 10 different immobilized oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

113.

19. The system of claim 11, wherein there are at least 50 different oligonucleotides, each at a distinct location on the array, and each of the at least 50 different immobilized oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

113.

20. The system of claim 11, wherein the ecosystem comprises water.

23. The system of claim 12, wherein at least one of the PCR primers comprises the sequence as set forth in any one of SEQ ID NO:

114-SEQ. ID NO;

316.

29. The system of claim 11, wherein the plurality of oligonucleotides are derived from at least one of a prokaryotic ribosomal DNA sequence that corresponds to nucleotides 160-250 of the prokaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO:

320 or nucleotides 430-510 of a prokaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

321, or from at least one of a eukaryotic ribosomal DNA sequences that correspond to nucleotides 600 to 800 of a eukaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO;

325;

or nucleotides 1000 to 1100 of the eukaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

327.

32. The system of claim 11, wherein each oligonucleotide has a sequence that is specific to a single, predetermined microbial operational taxonomic unit and wherein an operational taxonomic unit is comprised of sequences having at least 97.5% identity to each other.

21. A system for monitoring water quality comprising an array, wherein the array comprises a plurality of oligonucleotides covalently immobilized at known locations of the array, and wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit;

and

a plurality of oligonucleotides having a detectable label covalently attached thereto for hybridizing to the array wherein at least one of the detectably labeled oligonucleotides comprises the sequence as set forth in any one of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

22. The system of claim 21, wherein the system further comprises a detectably labeled oligonucleotide having the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

25, or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99, or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

25, or SEQ ID NO;

27-SEQ ID NO;

31, or SEQ ID NO;

33-SEQ ID NO;

39, or SEQ ID NO;

41-SEQ ID NO;

97, or SEQ ID NO;

99, or SEQ ID NO;

101-SEQ ID NO;

105, or SEQ ID NO;

107-SEQ ID NO;

113.

24. The system of claim 21, wherein there are at least 2 different oligonucleotides, each at a distinct location on the array, and each of the at least 2 different immobilized oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

5-SEQ ID NO;

113, or the complement of SEQ ID NO;

5-SEQ ID NO;

113.

25. The system of claim 21, wherein at least three of the plurality of detectably labeled oligonucleotides comprises the sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

26. The system of claim 21, wherein at least two of the plurality of detectably labeled oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

27. The system of claim 21, wherein at least four of the plurality of detectably labeled oligonucleotides individually comprises the sequence as set forth in any one of SEQ ID NO:

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106, or the complement of SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, SEQ ID NO;

100 or SEQ ID NO;

106.

33. The system of claim 21, wherein the plurality of oligonucleotides immobilized at known locations of the array are derived from at least one of a prokaryotic ribosomal DNA sequence that corresponds to nucleotides 160-250 of the prokaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO:

320 or nucleotides 430-510 of a prokaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

321, or from at least one of a eukaryotic ribosomal DNA sequences that correspond to nucleotides 600 to 800 of a eukaryotic small subunit ribosomal DNA V2 region as set forth in SEQ ID NO;

325;

or nucleotides 1000 to 1100 of the eukaryotic small subunit ribosomal DNA V4 region as set forth in SEQ ID NO;

327.

34. The system of claim 21, wherein the detectable label comprises one of a radionucleotide, a biotinylated nucleotide or a fluorescein-labeled nucleotide.