Arrays of nucleic acid probes on biological chips Number:7,115,364 from the United States Patent and Trademark Office (PTO) owispatent The invention provides chips of immobilized probes, and methods employing the chips, for comparing a reference polynucleotide sequence of known sequence with a target sequence showing substantial similarity with the reference sequence, but differing in the presence of e.g., mutations.<H biological, nucleic, Arrays, of, nucle, 7115364.html, Patent, pto, 7115364

Title: Arrays of nucleic acid probes on biological chips

Abstract: The invention provides chips of immobilized probes, and methods employing the chips, for comparing a reference polynucleotide sequence of known sequence with a target sequence showing substantial similarity with the reference sequence, but differing in the presence of e.g., mutations.

Patent Number: 7,115,364 Issued on 10/03/2006 to Chee, et al.

Inventors: Chee; Mark (Palo Alto, CA), Cronin; Maureen T. (Los Altos, CA), Fodor; Stephen P. A. (Palo Alto, CA), Gingeras; Thomas R. (Santa Clara, CA), Huang; Xiaohua C. (Mountain View, CA), Hubbell; Earl A. (Mountain View, CA), Lipshutz; Robert J. (Palo Alto, CA), Lobban; Peter E. (Palo Alto, CA), Miyada; Charles Garrett (Sunnyvale, CA), Morris; Macdonald S. (San Jose, CA), Shah; Nila (Saratoga, CA), Sheldon; Edward L. (San Diego, CA)
Assignee: Affymetrix, Inc. (Santa Clara, CA)

Appl. No.: 08/510,521

Filed: August 2, 1995

Current U.S. Class: 435/6 ; 422/50; 422/68.1; 536/24.3

Current International Class: C12Q 1/68 (20060101); C07H 21/04 (20060101); G01N 33/50 (20060101); G01N 33/53 (20060101)

Field of Search: 422/50,68.1 435/5,6,810,91.2 436/501 536/23.1,24.1,24.3-24.33,23.2,25.3 935/77,78

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Primary Examiner: Moran; Marjorie A.
Assistant Examiner: Miller; Marina
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part PCT/US94/12305, filed Oct. 26, 1994, which is a continuation-in-part of U.S. Ser. No. 08/284,064, filed Aug. 2, 1994 (now abandoned), which is a continuation-in-part of U.S. Ser. No. 08/143,312 (now abandoned), filed Oct. 26, 1993, each of which is incorporated by reference in its entirety for all purposes.

Claims

What is claimed is:

1. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence.

2. The array of claim 1, wherein the reference sequence is from an hMLH1 gene.

3. The array of claim 1, wherein the reference sequence is from an MSH2 gene.

4. An array of nucleic acid probes immobilized on a solid support, the array comprising at least first, second and third probe groups, each group comprising a first and second sets of probes as defined by claim 1; wherein each probe in the first probe set from the first group is exactly complementary to a subsequence of a first reference sequence and each probe in the first probe set from the second group is exactly complementary to a subsequence from a second reference sequence; and each probe in the first probe set from the third group is exactly complementary to as subsequence of a third reference sequence; wherein the first, second and third reference sequences are from a p53 gene, an hMLH1 gene, and an MSH2 gene, respectively.

5. An array of nucleic acid probes immobilized on a solid support, the array comprising at least four sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) second, third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe in the second, third and fourth probe sets being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets; provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence.

6. The array of claim 5, wherein the first probe set has at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in a reference sequence.

7. The array of claim 5, wherein the first probe set has at least 50 interrogation positions respectively corresponding to each of 50 contiguous nucleotides in a reference sequence.

8. The array of claim 5, wherein the probes are linked to the support via a spacer.

9. The array of claim 5, wherein the segment in each probe of the first probe set that is exactly complementary to the subsequence of the reference sequence is 9 21 nucleotides.

10. The array of claim 5, wherein each probe of the first probe set consists of the segment that is exactly complementary to the subsequence of the reference sequence.

11. The array of claim 5, wherein the probes in the second, third and fourth probe sets are identical to the corresponding probe from the first probe set except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.

12. An array of nucleic acid probes immobilized on a solid support, the array comprising at least four sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) second, third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe in the second, third and fourth probe sets being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets; (3) a fifth probe set comprising a probe for each interrogation position in the first probe set, each probe in the fifth probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is deleted in the corresponding probe from the fifth probe set provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U.

13. An array of nucleic acid probes immobilized on a solid support, the array comprising at least four sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) second, third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe in the second, third and fourth probe sets being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets; (3) a fifth probe set comprising a probe for each interrogation position in the first probe set, each probe in the fifth probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that an additional nucleotide is inserted adjacent to the one interrogation position in the corresponding probe from the first probe set.

14. An array of nucleic acid probes immobilized on a solid support, the array comprising at least four sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) second, third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe in the second, third and fourth probe sets being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets; provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; wherein the array has between 100 and 100,000 probes.

15. An array of nucleic acid probes immobilized on a solid support, the array comprising at least one pair of first and second probe groups, each group comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; wherein each probe in the first probe set from the first group is exactly complementary to a subsequence of a first reference sequence and each probe in the first probe set from the second group is exactly complementary to a subsequence from a second reference sequence and the second reference sequence is a mutated form of the first reference sequence.

16. The array of claim 15, wherein each group further comprises third and fourth probe sets, each comprising a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.

17. The array of claim 16 that comprises at least forty pairs of first and second probe groups, wherein the probes in the first probe sets from the first groups of the forty pairs are exactly complementary to subsequences from forty respective first reference sequences.

18. An array of probes immobilized to a solid support comprising two blocks of probes, each block as defined by claim 17, a first block comprising a wildtype probe comprising a segment exactly complementary to a subsequence of a first reference sequence and a second block comprising a wildtype probe comprising a segment exactly complementary to a subsequence of a second reference sequence.

19. The array of claim 18, comprising at least 10 100 blocks of probes, each comprising a wildtype probe comprising a segment exactly complementary to a subsequence of at least 10 100 respective reference sequences.

20. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: hybridizing the target sequence to the array of claim 15; determining which probes in the first group, relative to one another, hybridize to the target sequence, the relative specific binding of the probes indicating whether the target sequence is the same or different from the first reference sequence; determining which probes in the second group, relative to one another, hybridize to the target sequence, the relative specific binding of the probes indicating whether the target sequence is the same or different from the second reference sequence.

21. The method of claim 20, wherein the hybridizing step comprising hybridizing the target sequence and a second target sequence to the array, and the relative specific binding of the probes from the first group indicates that the target is identical to the first reference sequence, and the relative specific binding of the probes from the second group indicates that the second target sequence is identical to the second reference sequence.

22. The method of claim 21, wherein the first and second target sequences are heterozygous alleles.

23. A block of nucleic acid probes immobilized on a solid support, the array comprising: a wildtype probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment having a plurality of interrogation positions respectively corresponding to a plurality of nucleotides in the reference sequence, for each interrogation position, three mutant probes, each identical to a sequence comprising the wildtype probe or a subsequence of at least six nucleotides thereof including the plurality of interrogation positions, except in the interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe; provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; wherein the reference sequence is from a gene having a variant form associated with development of cancer, a pathogenic microorganism, a biotransformation gene, or a gene associated with a hereditary disorder.

24. The array of claim 23, wherein the segment of the wildtype probe comprises 3 20 interrogation positions corresponding to 3 20 respective nucleotides in the reference sequence.

25. An array of nucleic acid probes immobilized on a solid support, the array comprising at least four probes: a first probe comprising first and second segments, each of at least three nucleotides and each exactly complementary to first and second subsequences of a reference sequence, the segments including at least one interrogation position corresponding to a nucleotide in the reference sequence, wherein either (1) the first and second subsequences are noncontiguous, or (2) the first and second subsequences are contiguous and the first and second segments are inverted relative to the complement of the first and second subsequences in the reference sequence; second, third and fourth probes, identical to a sequence comprising the first probe or a subsequence thereof comprising at least three nucleotides from each of the first and second segments, except in an interrogation position, which differs in each of the probes; provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U.

26. The array of any one of claims, 1, 5, 15, 23 and 25, wherein the reference sequence is from a gene having a variant form associated with development of cancer.

27. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U; wherein the reference sequence is from a p53 gene.

28. The array of claim 27, wherein the first probe set has at least 60 interrogation positions corresponding to at least 60 contiguous nucleotides from exon 6 of the p53 gene.

29. An array of nucleic acid probes immobilized on a solid support, the array comprising at least a set of four probes, each of the probes comprising a segment of at least 7 nucleotides that is exactly complementary to a subsequence from a reference sequence, except that the segment may or may not be exactly complementary at two interrogation positions, wherein: the first interrogation position is occupied by a different nucleotide in each of the four probes, the second interrogation position is occupied by a different nucleotide in each of the four probes, in first and second probes, the segment is exactly complementary to the subsequence, except at not more than one of the interrogation positions, and in third and fourth probes, the segment is exactly complementary to the subsequence, except at both of the interrogation positions, provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U.

30. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing a sample comprising the target nucleic acid to an array of nucleic acid probes immobilized on a solid support, the array comprising a set of probes comprising: a first probe comprising a segment of at least 7 nucleotides exactly complementary to a subsequence of a reference sequence except at one or two positions, the segment including an interrogation position not at the one or two positions; second, third and fourth mutant probes, each identical to a sequence comprising the wildtype probe or a subsequence thereof including the interrogation position and the one or two positions, except in the interrogation position, which is occupied by a different nucleotide in each of the four probes; provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U.

31. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing a sample comprising the target nucleic acid to an array of nucleic acid probes immobilized on a solid support, the array comprising: (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence; (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein, the probes in the first probe set have at least three interrogation positions respectively corresponding to each of at least three nucleotides in the reference sequence, and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence and (b) determining which probes, relative to one another, in the first and second probe sets specifically bind to the target nucleic acid, the relative specific binding of corresponding probes in the first and second probe sets indicating whether a nucleotide in the target sequence is the same or different from the corresponding nucleotide in the reference sequence.

32. The method of claim 31, wherein the array further comprises third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe in the second, third and fourth probe sets being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets; and the determining step comprises determining which probes, relative to one another, in the first, second, third and fourth probe sets specifically bind to the target nucleic acid, the relative specific binding of corresponding probes in the first, second, third and fourth probe sets indicating whether a nucleotide in the target sequence is the same or different from the corresponding nucleotide in the reference sequence.

33. The method of claim 32, wherein the determining comprises: (1) comparing the relative specific binding of four corresponding probes from the first, second, third and fourth probe sets; (2) assigning a nucleotide in the target sequence as the complement of the interrogation position of the probe having the greatest specific binding; (3) repeating (1) and (2) by comparing the relative specific binding of a further four corresponding probes from the first, second, third and fourth probe sets until each nucleotide of interest in the target sequence has been assigned.

34. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing a sample comprising the target nucleic acid to an array of nucleic acid probes immobilized on a solid support, the array comprising: (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence; (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein, the probes in the first probe set have at least three interrogation positions respectively corresponding to each of at least three nucleotides in the reference sequence, and: (b) comparing the relative specific binding of two corresponding probes from the first and second probe sets; (c) assigning a nucleotide in the target sequence as the complement of the interrogation position of the probe having the greater specific binding; (d) repeating (1) and (2) by comparing the relative specific binding of a further two corresponding probes from the first and second probe sets until each nucleotide of interest in the target sequence has been assigned.

35. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing the target nucleic acid to an array of probes immobilized on a solid support, the array comprising: a wildtype probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment having a plurality of interrogation positions respectively corresponding to a plurality of nucleotides in the reference sequence, wherein the reference sequence is from a gene having a variant form associated with development of cancer; for each interrogation position, three mutant probes, each identical to a sequence comprising the wildtype probe or a subsequence of at least six nucleotides thereof including the plurality of interrogation positions, except in the interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe; (b) for each interrogation position, (1) comparing the relative specific binding of the three mutant probes and the wildtype probe; (2) assigning a nucleotide in the target sequence as the complement of the interrogation position of the probe having the greatest specific binding.

36. The method of claim 35, wherein the target sequence has an undetermined substitution relative to the reference sequence, and the method assigns a nucleotide to the substitution.

37. The method of claim 36, wherein: the hybridizing step comprises hybridizing the target nucleic acid and a second target nucleic acid and the determining step comprises determining which probes, relative to one another, in the array bind specifically to the target nucleic acid or the second target nucleic acid, the relative specific binding of the probes indicating whether the target sequence is the same or different from the reference sequence and whether the second target sequence is the same or different from the reference sequence.

38. The method of claim 37, wherein the target sequence has a label and the second target sequence has a second label different from the label.

39. The method of claim 38, wherein undetermined first and second proportions of the first and second target sequences are hybridized to the array and the specific binding indicates the proportions.

40. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing the reference sequence to an array of nucleic acid probes immobilized on a solid support, the array comprising; (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of the reference sequence except in an interrogation position; (2) a second probe set comprising a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence; and (b) determining which probes in the first and second probe sets, relative to one another, in the array bind specifically to the reference sequence; (c) hybridizing a target sequence to the array; (d) determining which probes, relative to one another, in the array bind specifically to the target sequence; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence, wherein the relative specific binding of the probes to the reference and the target sequence indicates whether the reference sequence is the same or different from the target sequence.

41. The method of any one of claims 31 40, wherein the reference sequence is from a gene having a variant form associated with development of cancer.

42. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence; wherein the reference sequence is from a gene from an HIV virus.

43. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence, wherein the reference sequence is from a CFTR gene.

44. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U and wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence, wherein the reference sequence is from a mitochondrial genome.

45. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a probe for each interrogation position in the first probe set, each probe in the second probe set being identical to a sequence comprising a corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein the probes in the first probe set collectively have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence; provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U, the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence, and wherein the reference sequence is from a biotransformation gene.

46. An array of any one of claims 42 45, wherein the reference sequence is at least 50 bases, and the first probe set comprises overlapping probes spanning the reference sequence.

47. A method of comparing a target sequence with a reference sequence, the method comprising: identifying variants of a reference sequence differing from the reference sequence in at least one nucleotide; assigning each variant a designation, providing an array of pools of probes, each pool occupying a separate cell of the array, wherein each pool comprises a plurality of different probes, the different probes comprising different segments exactly complementary to respective different variant sequences assigned a particular designation, contacting the array with a target sequence comprising a variant of the reference sequence; determining the relative hybridization intensities of the pools in the array to the target sequence; determining the target sequence from the relative hybridization intensities of the pools.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides arrays of oligonucleotide probes immobilized in microfabricated patterns on chips for analyzing molecular interactions of biological interest. The invention therefore relates to diverse fields impacted by the nature of molecular interaction, including chemistry, biology, medicine, and medical diagnostics.

2. Description of Related Art

Oligonucleotide probes have long been used to detect complementary nucleic acid sequences in a nucleic acid of interest (the "target" nucleic acid). In some assay formats, the oligonucleotide probe is tethered, i.e., by covalent attachment, to a solid support, and arrays of oligonucleotide probes immobilized on solid supports have been used to detect specific nucleic acid sequences in a target nucleic acid. See, e.g., PCT patent publication Nos. WO 89/10977 and 89/11548. Others have proposed the use of large numbers of oligonucleotide probes to provide the complete nucleic acid sequence of a target nucleic acid but failed to provide an enabling method for using arrays of immobilized probes for this purpose. See U.S. Pat. Nos. 5,202,231 and 5,002,867 and PCT patent publication No. WO 93/17126.

The development of VLSIPS.TM. technology has provided methods for making very large arrays of oligonucleotide probes in very small areas. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092, each of which is incorporated herein by reference. U.S. patent application Ser. No. 08/082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific nucleotide sequence.

Microfabricated arrays of large numbers of oligonucleotide probes, called "DNA chips" offer great promise for a wide variety of applications. New methods and reagents are required to realize this promise, and the present invention helps meet that need.

SUMMARY OF THE INVENTION

The invention provides several strategies employing immobilized arrays of probes for comparing a reference sequence of known sequence with a target sequence showing substantial similarity with the reference sequence, but differing in the presence of, e.g., mutations. In a first embodiment, the invention provides a tiling strategy employing an array of immobilized oligonucleotide probes comprising at least two sets of probes. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. A second probe set comprises a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets. The probes in the first probe set have at least two interrogation positions corresponding to two contiguous nucleotides in the reference sequence. One interrogation position corresponds to one of the contiguous nucleotides, and the other interrogation position to the other.

In a second embodiment, the invention provides a tiling strategy employing an array comprising four probe sets. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. Second, third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set. The probes in the second, third and fourth probe sets are identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets. The first probe set often has at least 100 interrogation positions corresponding to 100 contiguous nucleotides in the reference sequence. Sometimes the first probe set has an interrogation position corresponding to every nucleotide in the reference sequence. The segment of complementarity within the probe set is usually about 9 21 nucleotides. Although probes may contain leading or trailing sequences in addition to the 9 21 sequences, many probes consist exclusively of a 9 21 segment of complementarity.

In a third embodiment, the invention provides immobilized arrays of probes tiled for multiple reference sequences. One such array comprises at least one pair of first and second probe groups, each group comprising first and second sets of probes as defined in the first embodiment. Each probe in the first probe set from the first group is exactly complementary to a subsequence of a first reference sequence, and each probe in the first probe set from the second group is exactly complementary to a subsequence of a second reference sequence. Thus, the first group of probes are tiled with respect to a first reference sequence and the second group of probes with respect to a second reference sequence. Each group of probes can also include third and fourth sets of probes as defined in the second embodiment. In some arrays of this type, the second reference sequence is a mutated form of the first reference sequence.

In a fourth embodiment, the invention provides arrays for block tiling. Block tiling is a species of the basic tiling strategies described above. The usual unit of a block tiling array is a group of probes comprising a wildtype probe, a first set of three mutant probes and a second set of three mutant probes. The wildtype probe comprises a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence. The segment has at least first and second interrogation positions corresponding to first and second nucleotides in the reference sequence. The probes in the first set of three mutant probes are each identical to a sequence comprising the wildtype probe or a subsequence of at least three nucleotides thereof including the first and second interrogation positions, except in the first interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe. The probes in the second set of three mutant probes are each identical to a sequence comprising the wildtype probes or a subsequence of at least three nucleotides thereof including the first and second interrogation positions, except in the second interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.

In a fifth embodiment, the invention provides methods of comparing a target sequence with a reference sequence using arrays of immobilized pooled probes. The arrays employed in these methods represent a further species of the basic tiling arrays noted above. In these methods, variants of a reference sequence differing from the reference sequence in at least one nucleotide are identified and each is assigned a designation. An array of pooled probes is provided, with each pool occupying a separate cell of the array. Each pool comprises a probe comprising a segment exactly complementary to each variant sequence assigned a particular designation. The array is then contacted with a target sequence comprising a variant of the reference sequence. The relative hybridization intensities of the pools in the array to the target sequence are determined. The identity of the target sequence is deduced from the pattern of hybridization intensities. Often, each variant is assigned a designation having at least one digit and at least one value for the digit. In this case, each pool comprises a probe comprising a segment exactly complementary to each variant sequence assigned a particular value in a particular digit. When variants are assigned successive numbers in a numbering system of base m having n digits, n.times.(m-1) pooled probes are used to assign each variant a designation.

In a sixth embodiment, the invention provides a pooled probe for trellis tiling, a further species of the basic tiling strategy. In trellis tiling, the identity of a nucleotide in a target sequence is determined from a comparison of hybridization intensities of three pooled trellis probes. A pooled trellis probe comprises a segment exactly complementary to a subsequence of a reference sequence except at a first interrogation position occupied by a pooled nucleotide N, a second interrogation position occupied by a pooled nucleotide selected from the group of three consisting of (1) M or K, (2) R or Y and (3) S or W, and a third interrogation position occupied by a second pooled nucleotide selected from the group. The pooled nucleotide occupying the second interrogation position comprises a nucleotide complementary to a corresponding nucleotide from the reference sequence when the second pooled probe and reference sequence are maximally aligned, and the pooled nucleotide occupying the third interrogation position comprises a nucleotide complementary to a corresponding nucleotide from the reference sequence when the third pooled probe and the reference sequence are maximally aligned. Standard IUPAC nomenclature is used for describing pooled nucleotides.

In trellis tiling, an array comprises at least first, second and third cells, respectively occupied by first, second and third pooled probes, each according to the generic description above. However, the segment of complementarity, location of interrogation positions, and selection of pooled nucleotide at each interrogation position may or may not differ between the three pooled probes subj