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US 7,115,364 B1Granted
Arrays of nucleic acid probes on biological chips
AssigneeAffymetrix, Inc.
InventorsMark Chee, Maureen T. Cronin, Stephen P. A. Fodor, Thomas R. Gingeras, Xiaohua C. Huang, Earl A. Hubbell, Robert J. Lipshutz, Peter E. Lobban, Charles Garrett Miyada, Macdonald S. Morris, Nila Shah, Edward L. Sheldon
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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.
Claims47
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 more elements
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.
1 more element
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.
1 more element
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.
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.
1 more element
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.
2 more elements
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.
1 more element
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.
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.
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.
2 more elements
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.
2 more elements
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.
1 more element
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.
2 more elements
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.
4 more elements
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.
3 more elements
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.
5 more elements
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.
2 more elements
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.
2 more elements
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.
2 more elements
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.
2 more elements
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.
3 more elements
Dependencies are inferred from the claim text.