Universal microarray system

• US 20030003484A1
• Filed: 05/09/2002
• Published: 01/02/2003
• Est. Priority Date: 05/09/2001
• Status: Active Grant

• Current Assignee: Foodchain ID Testing Incorporated
• Original Assignee: Genetic ID NA Incorporated
• Inventors: Fagan, John
• Granted Patent: US 7,138,506 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

The present invention provides a simple, cost-effective, universal method for determining and/or quantifying differences in nucleic acid levels between two or more test mixtures without prior knowledge of the sequence of the nucleic acids of interest. The method involves providing a universal microarray containing a plurality of spots, where each spot contains a plurality, or pool, of different oligonucleotide probes having at least three distinct portions: a universal sequence portion, a short central variable “wobble” sequence portion, and a unique sequence portion. A set of probes is synthesized such that the universal sequence portion is the same for every probe, and all possible permutations of the wobble sequence and unique sequence portions are represented in approximately equal concentrations in the set. The probes are pooled on the universal microarray such that probes in a given spot have the same unique portion and every permutation of the short wobble, while probes on different spots have different unique portions. Primers complementary to the universal and wobble portions of the probes are used to synthesize, for example, cDNA from an mRNA preparation. The cDNA is hybridized to the universal microarray, and through the use of differential labeling, is identified and/or quantified.

Claims

1. A universal microarray comprising a solid substrate and a plurality of oligonucleotide probes bound to a plurality of spots on said substrate, said plurality of oligonucleotide probes having the form:

B-C-D

wherein

a) portion B is a unique z-mer comprising from about 5 to about 8 nucleotide bases such that all 4^z permutations of A, G, C, and T are represented in said plurality of oligonucleotide probes and only one of said 4^z permutations is present in each spot;

b) portion C is a variable x-mer comprising at least 1 nucleotide base such that all 4^x permutations of A, G, C, and T are represented in said plurality of oligonucleotide probes and all 4^x permutations are present in every spot in about equal concentrations;

c) portion D is a universal n-mer comprising from about 5 to about 8 nucleotide bases that is the same for every probe.

2. The microarray of claim 1 wherein portions B, C, and D are joined in the 5′

- to -3′

direction or the 3′

- to -5′

direction, respectively.

3. The microarray of claim 1 wherein portions B, C and D are joined in the 5′

- 3′

direction.

4. The microarray of claim 1 wherein z is about 5 bases.

5. The microarray of claim 1 wherein z is about 8 bases.

6. The microarray of claim 1 wherein portion B is a hexamer.

7. The microarray of claim 1 wherein n is about 5 bases.

8. The microarray of claim 1 wherein n is about 8 bases.

9. The microarray of claim 1 wherein portion D is a hexamer.

10. The microarray of claim 1 wherein x is about 1 to about 5 bases.

11. The microarray of claim 1 wherein portion C is a dimer.

12. The microarray of claim 1 wherein each oligonucleotide probe in said plurality of oligonucleotide probes further comprises a portion E that is a t-mer of about 1 to about 4 nucleotides adjacent to portion B or portion D at either the 5′

end or the 3′

end of each oligonucleotide probe wherein the sequence of portion E is variable within any given spot such that all possible 4^t permutations of A, G, C, and T are represented within any given spot for each permutation of B-C-D within that spot.

13. The microarray of any one of claims 1-12 wherein the plurality of probes further comprises a linker group A, for attachment of a probe to a substrate, bound at either the 5′

or 3′

end of each of said probes.

14. The microarray of claim 1 wherein said substrate is chosen from the group consisting of polymers, glasses, semiconductors, papers, metals, gels and hydrogels.

15. The microarray of claim 14 wherein said substrate is further modified or coated to contain reactive functional groups.

16. The microarray of claim 14 wherein said substrate is glass.

17. The microarray of claim 16 wherein said glass is coated with a polymer or metal.

18. The microarray of claim 15 wherein said substrate or said coating on said substrate is gold or thiolated gold.

19. The microarray of claim 1 wherein the universal n-mer comprises about 40% to abut 70% GC.

20. The microarray of claim 1 wherein the universal n-mer comprises greater than about 50% GC.

21. The microarray of claim 1 wherein the number of spots is about 4096.

22. The microarray of claim 1 wherein said substrate is segmented to form groups of spots.

23. The microarray of claim 22 wherein the number of spots per group is less than about 40.

24. The microarray of claim 22 wherein said groups are formed based on a physical property of the probes.

25. The microarray of claim 24 wherein said physical property is the theoretical melting temperature of the probes and their complements.

27. A set of primers having the form

F-G

wherein F and G are the same length as and complementary to the universal n-mer and variable x-mers, respectively, of the probes of claim 1 such that all possible 4^x permutations of F-G are represented in said set of primers.

28. A set of primers having the form

F-G

wherein F and G are complementary to the universal hexamer and variable dimer, respectively, of the probes of claim 1 such that all possible 4² permutations of F-G are represented in said set of primers.

29. A method for determining relative gene expression between 2 or more test mixtures comprising:

a) providing a first set of a plurality of oligonucleotide primers having the form;

F-G

wherein portion F is a universal n-mer comprising from about 5 to about 8 nucleotide bases and portion G is a variable x-mer comprising at least 1 nucleotide base such that all 4^x permutations of A, G, C, and T are represented in said primer set;

b) separately contacting each of the populations of mRNA derived from each of the 2 or more test mixtures to each of the members of said set of primers to generate 4^x primed mRNA populations for each test mixture;

c) segregating the primed mRNA populations into one or more subsets of mRNA populations;

d) separately synthesizing cDNA populations from each of the primed mRNA populations in each subset of mRNA populations by reverse transcription;

e) recovering each of said synthesized cDNA populations with said primers attached thereto to obtain one or more cDNA subsets corresponding to the subsets of the primed mRNA populations from which they were derived;

f) differentially labeling each member of a subset of cDNA populations.

g) providing a microarray of claim 1 for each subset of cDNA populations, such that portions C and D of the probes of said microarray are complementary to portions G and F, respectively, of the primer set used to synthesize the cDNA populations;

h) contacting one or more spots on the microarray with the differentially labeled members of a subset of cDNA populations such that each spot is contacted with all of the differentially labeled members of a subset of cDNA populations under conditions such that complementary cDNA and probe sequences hybridize;

i) repeating steps f-h for each subset of cDNA populations such that each subset of cDNA populations contacts a microarray not previously contacted with any other subset of cDNA populations;

j) detecting the signal generated from every spot on every array;

k) determining relative gene expression of the test mixtures by comparing the signal from a spot on one array to the corresponding spot on every other array wherein the probes on corresponding spots comprise the same unique z-mer, or comparing the signal from different labels on a single spot.

30. The method of claim 29 wherein said members of a cDNA subset are pooled prior to said step h).

31. The method of claim 29 wherein the amount of cDNA in each cDNA population is normalized relative to every other population prior to step h).

32. The method of claim 29 wherein after said step of recovering, the concentration of cDNA in each of said labeled cDNA populations is normalized such that the total amount of cDNA contacting the spots in the microarray is about equal for each of said labeled cDNA populations.

33. The method of claim 29 wherein said step of labeling occurs during synthesis of the cDNA populations.

34. The method of claim 33 wherein at least one of the dNTPs used for reverse transcription is present in a form that terminates cDNA synthesis.

35. The method of claim 34 wherein the dNTP that terminates cDNA population synthesis is labeled.

36. The method of claim 34 wherein cDNA is terminated at about 50 to about 100 bases.

37. The method of claim 33 wherein at least one of the dNTPs used for reverse transcription is present in labeled and unlabeled forms.

38. The method of claim 29 wherein cDNA is labeled after its synthesis.

39. The method of claim 29 wherein said cDNA is labeled with a label chosen from the group consisting of dyes, particles, and radioactive substances.

40. The method of claim 29 wherein said cDNA is labeled with a dye chosen from the group consisting of fluorescent, chemiluminescent, bioluminescent, and electroluminescent dyes.

41. The method of claim 29 wherein said cDNA is labeled with a radioactive label.

42. The method of claim 29 wherein said cDNA is labeled with a particle chosen from the group consisting of glass, silica, polymer, metal, and semiconductor particles.

43. The method of claim 29 further comprising:

a) providing a second set of microarrays and a second set of complementary primers wherein the complementary universal n-mers of the probes and primers in the second set are different from the universal n-mers of the first set.

b) performing steps a-k of claim 29 using said second set of microarrays and primers.

44. The method of claim 43 wherein the probes and primers in the second set have a different G C content than the probes and primers of the first set.

45. The method of claim 43 further comprising:

a) providing a third set of microarrays and a third set of complementary primers wherein the complementary universal n-mers of the probes and primers in the third set are different from the universal n-mers of the first and second sets.

b) performing steps a-k of claim 29 using said third set of microarrays and primers.

46. The method of claim 45 wherein the probes and primers in the third set have a different G C content than the probes and primers of the first set and the probes and primers of the second set.

47. A kit for determining or measuring relative gene expression between 2 or more test mixtures comprising:

a) one or more microarrays of claim 1

b) one or more sets of primers, each primer having the form;

F-G

wherein F and G are the same length as and complementary to the universal n-mer and variable x-mers, respectively, of the probes on the one or more microarrays, such that all possible 4^x permutations of F-G are present in about equal concentrations in said set of primers.

48. The kit of claim 47 further comprising reagents for reverse transcription of mRNA populations from the 2 or more test mixtures to form cDNA populations.

49. The kit of claim 48 further comprising labels for differentially labeling the cDNA populations.

50. The kit of claim 49 further comprising instructions for binding cDNA to said microarrays and for determining relative gene expression based on said binding.

26. A universal microarray comprising a solid substrate and a plurality of oligonucleotide probes bound to a plurality of spots on said substrate, said plurality of oligonucleotide probes having the form:

B-C-D

wherein

a) portion B is a unique hexamer such that all 4⁶ permutations of A, G, C, and T are represented in said plurality of oligonucleotide probes and only one of said 4⁶ permutations is present in each spot;

b) portion C is a variable dimer such that all 4² permutations of A, G, C, and T are represented in said plurality of oligonucleotide probes and all 4² permutations are present in every spot in about equal concentrations;

c) portion D is a universal hexamer that is the same for every probe.