Methods and compositions for selecting tag nucleic acids and probe arrays

Claim 1

A method of selecting a set of tag nucleic acids with minimal cross hybridization to a nucleic acid, said method comprising providing a list of tag nucleic acids, and excluding nucleic acids from the list of tag nucleic acids which cross hybridize to a single complementary nucleic acid under stringent conditions, thereby providing a set of selected tag nucleic acids with minimal cross hybridization to the nucleic acid.

Claim 4

A method of any one of claims 1 to 3, wherein the method further comprises excluding tags which contain self-complementary regions from the list of tags, preferably regions of self complementarity greater than 4 nucleotides in length.

Claim 5

A method of any one of claims 1 to 4, wherein the tags are between 15 and 30 nucleotides in length or between 10 and 100 nucleotides in length, preferably 20 nucleotides in length.

Claim 6

A method of any one of claims 1 to 5, wherein the method further comprises selecting a complementary probe nucleic acid for tags in the selected tag set, wherein each tag sequence is complementary to one probe sequence, and the thermal binding stability between each tag and each complementary probe is substantially similar, preferably all of the tags having the same length and the same GC to AT ratio.

Claim 7

A method of any one of claims 1 to 6, wherein the method further comprises selecting a constant region subsequence shared by all tag nucleic acids, thereby determining the nucleotide position of variable nucleotides in the tags; optionally the method further comprising providing a set of probe nucleic acids by determining the complement to each variable nucleotide in each tag nucleic acid, and selecting a probe comprising a corresponding complementary nucleotide for each nucleotide in the variable tag sequence, which probe does not hybridize to the constant region of the tag nucleic acid, thereby providing a selected set of probes.

Claim 8

A method of any one of claims 1 to 7, wherein the method further comprises removing tag nucleic acids which have fewer than five, and preferably fewer than two, nucleotide differences when aligned for maximal sequence correspondence; preferably the total number of nucleotides in each of the selected sets being identical; the number of G+C nucleotides in each tag in the selected set being identical; and, the overall number of A+G nucleotides in each of the variable regions of the tags being even.

Claim 9

A method of any one of claims 1 to 8, wherein tags which contain 4 contiguous nucleotides selected from the group consisting of 4 X residues, 4 Y residues and 4 Z residues, are eliminated from the tag set, wherein X is selected from the group consisting of G and C, Y is selected from the group consisting of G and A, and Z is selected from the group consisting of A and T.

Claim 11

A method of labeling a composition, comprising associating a tag nucleic acid with the composition, wherein the tag nucleic acid is selected from a group of tag nucleic acids which do not cross-hybridize and which have a substantially similar T m .

Claim 15

A method of pre-selecting experimental probes in an oligonucleotide probe array, wherein the probes have substantially uniform hybridization properties and do not cross hybridize, comprising: selecting a ratio of G+C to A+T nucleotides shared by the experimental probes in the array; determining all possible 4 nucleotide subsequences for variable nucleic acids in the probes of the array; and excluding all probes from the array which contain prohibited 4 nucleotide subsequences, wherein 4 nucleotide subsequences are prohibited when the nucleotide subsequences are selected from the group consisting of self-complementary probes, A 4 probes, T 4 probes, [G,C] 4 probes, and probes complementary to constant region sub-sequences; optionally wherein the method further comprises selecting a length for the probes in the array, thereby providing selected length probes; selecting a constant region subsequence shared by all selected length probes in the array, thereby determining the nucleotide position of variable nucleic acids in the probes of the array; and providing that the overall number of A+G nucleotides in the probes of the array is even.

Claim 17

A method of detecting a plurality of nucleic acids in a sample, comprising (i) providing an array of experimental oligonucleotide probes, which probes do not cross hybridize under stringent conditions, wherein the ratio of G+C bases in each probe is substantially identical; wherein the probes of the array are arranged into probe sets in which each probe set comprises a homogeneous population of oligonucleotide probes; preferably the nucleic acids comprising tag sequences, which tag sequences bind to the probes of the array; (ii) hybridizing said array of oligonucleotides to the sample under stringent hybridization conditions, optionally the probes of the array specifically hybridizing to at least one nucleic acid in the sample; and (iii) detecting hybridization of the nucleic acids to the array of oligonucleotide probes.

Claim 20

A method of detecting a target nucleic acid comprising providing a population of nucleic acids to an array of oligonucleotide probes and monitoring hybridization of the test nucleic acids to the probes in the array, wherein the array of oligonucleotide probes comprises a plurality of experimental oligonucleotide probe sets attached to a solid substrate, wherein each experimental oligonucleotide probe set in the array hybridizes to a different target nucleic acid under stringent hybridization conditions; each oligonucleotide probe in the probe sets of the array comprises variable region; optionally the probes of the array comprising a constant region, wherein the variable region does not cross hybridize with the constant region under stringent hybridization conditions; and wherein the nucleic acid probes do not cross-hybridize in the array; preferably wherein the array also comprises a control probe, and wherein the method further comprises hybridizing a nucleic acid complementary to the control probe to the array.