Nucleic acid arrays to monitor water and other ecosystems

• US 7,214,492 B1
• Filed: 03/03/2005
• Issued: 05/08/2007
• 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: Henrich, Vincent C. III, Rublee, Parke A.
• Primary Examiner(s): Switzer; Juliet C.
• Assistant Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US11/071,849
• Time in Patent Office: 796 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence

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 method for monitoring an ecosystem of interest comprising:

(a) generating an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, wherein each location on the substrate comprises an oligonucleotide having a sequence derived from a single, predetermined microbial operational taxonomic unit (OTU), and wherein the array comprises at least one oligonucleotide having the sequence as set forth in SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

98, or SEQ ID NO;

100, or SEQ ID NO;

106, the complement of the sequence as set forth in SEQ ID NO;

25, 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;

(b) preparing a nucleic acid sample from the ecosystem of interest;

(c) hybridizing the nucleic acid sample to the array;

(d) measuring hybridization of the nucleic acid sample to the array; and

(e) correlating hybridization of the nucleic acid sample to the array with at least one parameter that comprises at least part of the ecosystem of interest.

2. The method of claim 1, wherein the ecosystem of interest comprises water.

3. The method of claim 2, wherein the at least one parameter comprises water quality.

4. The method of claim 1, wherein at least one of the immobilized oligonucleotides comprises a eukaryotic ribosomal DNA sequence and at least one of the immobilized oligonucleotides comprises a prokaryotic ribosomal DNA sequence.

5. The method of claim 1, wherein at least one immobilized oligonucleotide comprises the sequence as set forth in any one of SEQ ID NO:

5–

SEQ ID NO;

113, or the complement of any one of SEQ ID NO;

5–

SEQ ID NO;

113.

6. The method of claim 1, wherein there are at least 10 different immobilized oligonucleotides and each of the at least 10 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 any one of SEQ ID NO;

5–

SEQ ID NO;

113.

7. The method of claim 1, wherein there are at least 50 different immobilized oligonucleotides and each of the at least 50 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 any one of SEQ ID NO;

5–

SEQ ID NO;

113.

8. The method of claim 1, wherein the nucleic acid sample hybridized to the array comprises a plurality of rDNA sequences.

9. The method of claim 1, wherein the nucleic acid sample hybridized to the array is generated using polymerase chain reaction (PCR) primers derived at least in part from a small subunit (SSU) ribosomal DNA variable region.

10. The method of claim 9, wherein at least one of the PCR primers comprises the sequence identical to any one of SEQ ID NO:

114–

SEQ ID NO;

316.

11. The method of claim 1, wherein a single change in a pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in one or more parameters specific to at least part of the ecosystem of interest.

12. The method of claim 1, wherein a plurality of changes in a pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in one or more parameters specific to at least part of the ecosystem of interest.

18. The method of claim 1, wherein the plurality of immobilized oligonucleotides further comprises at least one oligonucleotide having the sequence as set forth in SEQ ID NO:

20, SEQ ID NO;

107, or SEQ ID NO;

109, or the complement of the sequence as set forth in SEQ ID NO;

20, SEQ ID NO;

107, or SEQ ID NO;

109.

19. The method of claim 1, wherein the plurality of immobilized oligonucleotides further comprises at least one oligonucleotide having the sequence as set forth in SEQ ID NO:

13, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

84, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

91, SEQ ID NO;

94, SEQ ID NO;

97, SEQ ID NO;

99, SEQ ID NO;

101, SEQ ID NO;

102, SEQ ID NO;

103, SEQ ID NO;

104, SEQ ID NO;

105, SEQ ID NO;

108, SEQ ID NO;

110, SEQ ID NO;

111, or SEQ ID NO;

112, or the complement of the sequence as set forth in SEQ ID NO;

13, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

84, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

91, SEQ ID NO;

94, SEQ ID NO;

97, SEQ ID NO;

99, SEQ ID NO;

101, SEQ ID NO;

102, SEQ ID NO;

103, SEQ ID NO;

104, SEQ ID NO;

105, SEQ ID NO;

108, SEQ ID NO;

110, SEQ ID NO;

111, or SEQ ID NO;

112.

13. A method for analyzing a pattern to evaluate the status of an ecosystem of interest comprising:

measuring hybridization of a nucleic acid sample isolated from the ecosystem of interest to an array of oligonucleotides immobilized at known locations on a substrate, wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU), and wherein the array comprises at least one oligonucleotide having the sequence as set forth in SEQ ID NO;

25, 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 the sequence as set forth in SEQ ID NO;

25, 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

correlating a pattern of hybridization of the nucleic acid sample to the array with at least one parameter that comprises at least part of the ecosystem of interest.

14. The method of claim 13, wherein a single change in the pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in one parameter that comprises at least part of the ecosystem of interest.

15. The method of claim 13, wherein a single change in the pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in a plurality of parameters that comprise at least part of the ecosystem of interest.

16. The method of claim 13, wherein a plurality of changes in the pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in one parameter that comprises at least part of the ecosystem of interest.

17. The method of claim 13, wherein a plurality of changes in the pattern of hybridization to the array for a second DNA sample isolated from the ecosystem of interest as compared to a first DNA sample isolated from the ecosystem of interest is associated with a change in a plurality of parameters that comprise at least part of the ecosystem of interest.

20. The method of claim 13, wherein at least one immobilized oligonucleotide comprises the sequence identical to any one of SEQ ID NO:

5–

SEQ ID NO;

113, or the complement of SEQ ID NO;

5- SEQ ID NO;

113.

21. The method of claim 13, wherein are at least 10 different immobilized oligonucleotides and each of the at least 10 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.

22. The method of claim 13, wherein there are at least 50 different immobilized oligonucleotides and each of the at least 50 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.

23. The method of claim 13, wherein the plurality of immobilized oligonucleotides further comprise at least one oligonucleotide having the sequence as set forth in SEQ ID NO:

20, SEQ ID NO;

107, SEQ ID NO;

109, or the complement of the sequence as set forth in SEQ ID NO;

20, SEQ ID NO;

107, or SEQ ID NO;

109.

24. The method of claim 13, wherein the plurality of immobilized oligonucleotides further comprise at least one oligonucleotide having the sequence as set forth in SEQ ID NO:

13, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

84, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

91, SEQ ID NO;

94, SEQ ID NO;

97, SEQ ID NO;

99, SEQ ID NO;

101, SEQ ID NO;

102, SEQ ID NO;

103, SEQ ID NO;

104, SEQ ID NO;

105, SEQ ID NO;

108, SEQ ID NO;

110, SEQ ID NO;

111, or SEQ ID NO;

112, or the complement of the sequence as set forth in SEQ ID NO;

13, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

84, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

91, SEQ ID NO;

94, SEQ ID NO;

97, SEQ ID NO;

99, SEQ ID NO;

101, SEQ ID NO;

102, SEQ ID NO;

103, SEQ ID NO;

104, SEQ ID NO;

105, SEQ ID NO;

108, SEQ ID NO;

110, SEQ ID NO;

11, or SEQ ID NO;

112.