Polynucleotides for use as tags and tag complements, manufacture and use thereof

• US 20050191625A1
• Filed: 01/25/2002
• Published: 09/01/2005
• Est. Priority Date: 01/25/2001
• Status: Active Grant

• Current Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Original Assignee: Tm Bioscience Corporation (DiaSorin S.p.A.)
• Inventors: Kobler, Daniel, Fieldhouse, Daniel
• Granted Patent: US 7,645,868 B2
• Time in Patent Office: Days
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc. A specific family of 1168 24mers is described.

Claims

1. A composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a following group of sequences,

2. A composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences having the numeric pattern of sequences set out in claim 1, wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

for any pair of sequences of the set;

M1≦

19, M2≦

17, M3≦

21, M4≦

18, and M5≦

20, where;

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score;

wherein

the score of an alignment is determined according to the equation (A×

m)−

(B×

mm) −

(C×

(og +eg))−

(D×

eg)), wherein;

for each of (i) to (iv);

 

(i) m=6, mm=6, og=0 and eg=6,

 

(ii) m=6, mm=6, og=5 and eg=1,

 

(iii) m=6, mm=2, og=5 and eg=1,and

 

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

3. A composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences having the numeric patterns set out in claim 1, wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

for any pair of sequences of the set;

M1≦

19, M17, M3≦

21, M4≦

18, and M5≦

20, where;

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score, wherein;

the score of an alignment is determined according to the equation 3A−

B−

3C−

D, wherein;

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignments.

4. A composition according to claim 1, wherein for the group of 24mer sequences in which 1=A, 2=T and 3=G, under a defined set of conditions in which the maximum degree of hybridization between a sequence and any complement of a different sequence of the group of 24mer sequences does not exceed 30% of the degree of hybridization between said sequence and its complement, for all said oligonucleotides of the composition, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the composition does not exceed 50% of the degree of hybridization of the oligonucleotide and its complement, and wherein when said maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 30% of the degree of hybridization between said sequence and its complement, the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 10 more preferably between 1 and up to 9, more preferably between 1 and up to 8 more preferably between 1 and up to 7 more preferably between 1 and up to 6. and more preferably between 1 and up to 5.

5. The composition of claim 2, wherein for the group of 24mer sequences in which 1=A, 2=T and 3=G under a defined set of conditions in which the maximum degree of hybridization between a sequence and any complement of a different sequence of the group of 24mer sequences does not exceed 30% of the degree of hybridization between said sequence and its complement for all said oligonucleotides of the composition the maximum degree of hybridization between an oliponucleotide and a complement of any other oliponucleotide of the composition does not exceed 50% of the degree of hybridization of the oligonucleotide and its complement, and wherein when said maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 30% of the degree of hybridization between said sequence and its complement, the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 10, more preferably between 1 and up to 9, more preferably between 1 and up to 8, more preferably between 1 and up to 7, more preferably between I and up to 6, and more preferably between 1 and up to 5.

6. The composition of claim 4 or 5, wherein the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 25%, more preferably does not exceed 20%, more preferably does not exceed 15%, more preferably does not exceed 10%, more preferably does not exceed 5%.

7. The composition of any of claim 4 or 5, wherein said defined set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C.

8. The composition of claim 4 or 5, wherein said defined set of conditions includes the group of 24mer sequences being covalently linked to beads.

9. The composition of claim 4 or 5, wherein, for the group of 24mers the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 15% of the degree of hybridization between said sequence and its complement and the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 9, and for all oligonucleotides of the set, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the set does not exceed 20% of the degree of hybridization of the oligonucleotide and its complement.

10. The composition of claim 4 or 5, wherein:

each 1 is one of A, T/U, G and C;

each 2 is one of A, T/U, G and C; and

each 3 is one of A, T/U, G and C; and

each of 1, 2 and 3 is selected so as to be different from all of the others of 1, 2 and 3.

11. The composition of claim 10, wherein 1 is A or T/U, 2 is A or T/U and 3 is G or C.

12. The composition of claim 11, wherein 1 is A, 2 is T/U and 3 is G.

13. The composition of claim 1 or 2, wherein each said oligonucleotide is from twenty-two to twenty-six bases in length, or from twenty-three to twenty-five.

14. The composition of claim 1 or 2, wherein each said oligonucleotide is of the same length as every other said oligonucleotide.

15. The composition of claim 14, wherein each said oligonucleotide is twenty-four bases in length.

16. The composition of claim 1 or 2, wherein no said oligonucleotide contains more than four contiguous bases that are identical to each other.

17. The composition of claim 1 or 2, wherein the number of G'"'"'s in each said oligonucleotide does not exceed L/4 where L is the number of bases in said sequence.

18. The composition of claim 1 or 2, wherein the number of G'"'"'s in each said oligonucleotide does not vary from the average number of G'"'"'s in all of the said oligonucleotides by more than one.

19. The composition of claim 1 or 2, wherein the number of G'"'"'s in each said oligonucleotide is the same as every other said oligonucleotide.

20. The composition of claim 19, wherein each said oligonucleotide is twenty-four bases in length and each said oligonucleotide contains 6 G'"'"'s.

21. The composition of claim 1 or 2, wherein, for each said nucleotide, there is at most six bases other than G between every pair of neighboring pairs of G'"'"'s.

22. The composition of claim 1 or 2, wherein at the 5′

-end of each said oligonucleotide at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonculeotide is a G.

23. The composition of claim 1 or 2, wherein at the 3′

-end of each said oligonucleotide at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonucleotide is a G.

24. The composition of claim 22, wherein at the 3′

-end of each said oligonucleotide at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonucleotide is a G.

25. The composition of claim 1 or 2, comprising ten, or twenty, or thirty, or forty, or fifty, or sixty, or seventy, or eighty, or ninety, or one hundred, or one hundred and ten, or one hundred and twenty, or one hundred and thirty, or one hundred and forty, or one hundred and fifty, or one hundred and sixty said molecules, or comprising one hundred and seventy said molecules, or comprising one hundred and eighty said molecules, or comprising one hundred and ninety said molecules, or comprising two hundred said molecules, or comprising two hundred and twenty said molecules, or comprising two hundred and forty said molecules, or comprising two hundred and sixty said molecules, or comprising two hundred and eighty said molecules, or comprising three hundred said molecules, or comprising four hundred said molecules, or comprising five hundred said molecules, or comprising six hundred said molecules, or comprising seven hundred said molecules, or comprising eight hundred said molecules, or comprising nine hundred said molecules, or comprising one thousand said molecules, or comprising eleven hundred said molecules.

26. A composition of claim 1 or 2, wherein each said molecule is linked to a solid phase support so as to be distinguishable from a mixture of other said molecules by hybridization to its complement.

27. The composition of claim 26, wherein each molecule is linked to a defined location on a said solid phase support, the defined location for each said molecule being different than the defined location for different other said molecules.

28. The composition of claim 27, wherein each said solid phase support is a microparticle and each said molecule is covalently linked to a different microparticle than each other different said molecule.

73. A composition according to claim 2, wherein any base is substituted by an analogue thereof.

74. A composition according to claim 2, wherein each said molecule comprises a tag complement.

75. A kit for sorting and identifying polynucleotides, the kit comprising one or more solid phase supports each having one or more spatially discrete regions, each such region having a uniform population of substantially identical tag complements covalently attached, and the tag complements each being selected from the set of oligonucleotides as defined in claim 1.

76. A kit according to claim 75, wherein there is a tag complement for each said oligonucleotide of a said composition.

77. A kit according to claim 75 or 76 wherein said one or more solid phase supports is a planar substrate and wherein said one or more spatially discrete regions is a plurality of spatially addressable regions.

78. A kit according to claim 75, wherein said one or more solid phase supports is a plurality of microparticles.

79. A kit according to claim 78, wherein said microparticles each have a diameter in the range of from 5 to 40 μ

m.

80. A kit according to claim 78 or 79, wherein each microparticle is spectrophotometrically unique from each other microparticle having a different oligonucleotide attached thereto.

81. A method of analyzing a biological sample comprising a biological sequence for the presence of a mutation or polymorphism at a locus of the nucleic acid, the method comprising:

(A) amplifying the nucleic acid molecule in the presence of a first primer having a 5′

-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements as defined in claim 74 to form an amplified molecule with a 5′

-end with a sequence complementary to the sequence of the tag;

(B) extending the amplified molecule in the presence of a polymerase and a second primer having 5′

-end complementary the 3′

-end of the amplified sequence, with the 3′

-end of the second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is;

(i) capable of incorporation during transciption by the polymerase onto the 3′

-end of a growing nucleotide strand;

(ii) causes termination of polymerization; and

(iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus of the amplified sequence;

(C) specifically hybridizing the second primer to a tag complement having the tag complement sequence of (A); and

(D) detecting the nucleotide derivative incorporated into the second primer in (B) so as to identify the base located at the locus of the nucleic acid.

82. A method of analyzing a biological sample comprising a plurality of nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule, the method comprising:

(A) amplifying the nucleic acid molecule in the presence of a first primer having a 5′

-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements as defined in claim 74 to form an amplified molecule with a 5′

-end with a sequence complementary to the sequence of the tag;

(B) extending the amplified molecule in the presence of a polymerase and a second primer having 5′

-end complementary the 3′

-end of the amplified sequence, the 3′

-end of the second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is;

(i) capable of incorporation during transciption by the polymerase onto the 3′

-end of a growing nucleotide strand;

(ii) causes termination of polymerization; and

(iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus of the amplified molecule;

(C) specifically hybridizing the second primer to a tag complement having the tag complement sequence of (A); and

(D) detecting the nucleotide derivative incorporated into the second primer in (B) so as to identify the base located at the locus of the nucleic acid;

wherein each tag of (A) is unique for each nucleic acid molecule and steps (A) and (B) are carried out with said nucleic molecules in the presence of each other.

83. A method of analyzing a biological sample comprising a plurality of double stranded complementary nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule, the method comprising:

(A) amplifying the double stranded molecule in the presence of a pair of first primers, each primer having an identical 5′

-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements as defined in claim 74 to form amplified molecules with 5′

-ends with a sequence complementary to the sequence of the tag;

(B) extending the amplified molecules in the presence of a polymerase and a pair of second primers each second primer having a 5′

-end complementary a 3′

-end of the amplified sequence, the 3′

-end of each said second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is;

(i) capable of incorporation during transciption by the polymerase onto the 3′

-end of a growing nucleotide strand;

(ii) causes termination of polymerization; and

(iii) capable of differential detection, one from the other;

(C) specifically hybridizing each of the second primers to a tag complement having the tag complement sequence of (A); and

(D) detecting the nucleotide derivative incorporated into the second primers in (B) so as to identify the base located at said locus;

wherein the sequence of each tag of (A) is unique for each nucleic acid molecule and steps (A) and (B) are carried out with said nucleic molecules in the presence of each other.

84. A method of analyzing a biological sample comprising a plurality of nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule, the method comprising:

(a) hybridizing the molecule and a primer, the primer having a 5′

-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements as defined in claim 74 and a 3′

-end extending to immediately adjacent the locus;

(b) enzymatically extending the 3′

-end of the primer in the presence of a plurality of nucleoside triphosphate derivatives each of which is;

(i) capable of enzymatic incorporation onto the 3′

-end of a growing nucleotide strand;

(ii) causes termination of said extension; and

(iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus;

(c) specifically hybridizing the extended primer formed in step (b) to a tag complement having the tag complement sequence of (a); and

(d) detecting the nucleotide derivative incorporated into the primer in step (b) so as to identify the base located at the locus of the nucleic acid molecule;

wherein each tag of (a) is unique for each nucleic acid molecule and steps (a) and (b) are carried out with said nucleic molecules in the presence of each other.

85. The method of claim 82, wherein each said derivative is a dideoxy nucleoside triphosphate.

86. The method of claim 84, wherein each respective complement is attached as a uniform population of substantially identical complements in a spacially discrete region on one or more said solid phase supports.

87. The method of claim 86, each said tag complement comprises a label, each such label being different for respective complements, and step (d) includes detecting the presence of the different labels for respective hybridization complexes of bound tags and tag complements.

88. The hybridized molecule and primer of step (A) of claim 84.

89. A method of determining the presence of a target suspected of being contained in a mixture, the method comprising the steps of:

(i) labelling the target with a first label;

(ii) providing a first detection moiety capable of specific binding to the target and including a first tag;

(iii) exposing a sample of the mixture to the detection moiety under conditions suitable to permit (or cause) said specific binding of the molecule and target;

(iv) providing a family of tag complements as defined in claim 74 wherein the family contains a first tag complement having a sequence complementary to that of the first tag;

(v) exposing the sample to the family of tag complements under conditions suitable to permit (or cause) specific hybridization of the first tag and its tag complement;

(vi) determining whether a said first detection moiety hybridized to a first said tag complement is bound to a said labelled target in order to determine the presence or absence said target in the mixture.

90. The method of claim 89, wherein said first tag complement is linked to a solid support at a specific location of the support and step (vi) includes detecting the presence the first label at said specified location.

91. The method of claim 89, wherein said first tag complement comprises a second label and step (vi) includes detecting the presence of the first and second labels in a hybridized complex of the moiety and the first tag complement.

92. The method of claim 89, wherein said target is selected from the group consisting of organic molecules, antigens, proteins, polypeptides, antibodies and nucleic acids.

93. The method of claim 92, wherein said target is an antigen and said first molecule is an antibody specific for said antigen.

94. The method of claim 93, wherein the antigen is a polypeptide or protein and the labelling step includes conjugation of fluorescent molecules, digoxigenin, biotinylation and the like.

95. The method of claim 94, wherein said target is a nucleic acid and the labelling step includes incorporation of fluorescent molecules, radiolabelled nucleotide, digoxigenin, biotinylation and the like.

29. A composition comprising a set of 150 molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

M1≦

19/24×

L1, M2≦

17/24×

L1, M3≦

21/24×

L1, M4≦

18/24×

L1, M5≦

20/24×

L1, where L1 is the length of the shortest sequence of the pair, where;

M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment of the pair of sequences;

M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of the pair of sequences of maximum score, wherein;

the score of an alignment is determined according to the equation (A×

m)−

(B×

mm)−

(C×

(og+eg))−

(D×

eg)), wherein;

for each of (i) to (iv);

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

31. The composition of claim 29 or 30, wherein each said sequence has up to fifty bases.

32. The composition of claim 31, wherein each said sequence is between sixteen and forty bases in length, or between sixteen and thirty-five bases in length, or between eighteen and thirty bases in length, or between twenty and twenty-eight bases in length, or between twenty-one and twenty-seven bases in length, or between twenty-two and twenty-six bases in length.

33. The composition of claim 29 or 30, wherein each said sequence is of the same length as every other said sequence.

34. The composition of claim 33, wherein each said sequence is twenty-four bases in length.

35. The composition of claim 29 or 30, wherein no said sequence contains more than four contiguous bases that are identical to each other.

36. The composition of claim 29 or 30, wherein the number of G'"'"'s in each said sequence does not exceed L/4 where L is the number of bases in said sequence.

37. The composition of claim 36, wherein the number of G'"'"'s in each said sequence does not vary from the average number of G'"'"'s in all of the sequences of the set by more than one.

38. The composition of claim 37, wherein the number of G'"'"'s in each said sequence is the same as every other sequence of the set.

39. The composition of claim 37, wherein each said sequence is twenty-four bases in length and each said sequence contains 6 G'"'"'s.

40. The composition of claim 29 or 30, wherein, for each said sequence, there is at most six bases other than G between every pair of neighboring pairs of G'"'"'s.

41. The composition of claim 29 or 30, wherein at the 5′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

42. The composition of claim 29 or 30, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

43. The composition of claim 41, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

44. The composition of claim 29 or 30, wherein under a defined set of conditions, the maximum degree of hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 30% of the degree of hybridization between said oligonucleotide and its complement, more preferably 20%, more preferably 15%, more preferably 10%, more preferably 6%.

45. The composition of claim 44, wherein said set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C., and the oligonucleotides are covalently linked to microparticles.

46. The composition of claim 45, wherein under said defined set of conditions, the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5.

47. The composition of claim 29 or 30, comprising one hundred and sixty said molecules, or comprising one hundred and seventy said molecules, or comprising one hundred and eighty said molecules, or comprising one hundred and ninety said molecules, or comprising two hundred said molecules, or comprising two hundred and twenty said molecules, or comprising two hundred and forty said molecules, or comprising two hundred and sixty said molecules, or comprising two hundred and eighty said molecules, or comprising three hundred said molecules, or comprising four hundred said molecules, or comprising five hundred said molecules, or comprising six hundred said molecules, or comprising seven hundred said molecules, or comprising eight hundred said molecules, or comprising nine hundred said molecules, or comprising one thousand said molecules.

48. A composition of claim 29 or 30, wherein each said molecule is linked to a solid phase support so as to be distinguishable from a mixture of other said molecules by hybridization to its complement.

49. The composition of claim 48, wherein each molecule is linked to a defined location on a said solid phase support, the defined location for each said molecule being different than the defined location for different other said molecules.

50. The composition of claim 49, wherein each said solid phase support is a microparticle and each said molecule is covalently to a different microparticle than each other different said molecule.

30. A composition comprising a set of 150 molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

M1≦

19, M2≦

17, M3≦

21, M4≦

18, and M5≦

20, where;

M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment of the pair of sequences;

M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of the pair of sequences of maximum score, wherein;

the score of a said alignment is determined according to the equation 3A−

B−

3C−

D, wherein;

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment.

51. A composition comprising one hundred and fifty minimally cross-hybridizing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide comprising a sequence of nucleotide bases for which, under a defined set of conditions, the maximum degree of hybridization between a said oligonucleotide and any complement of a different oligonucleotide does not exceed about 20% of the degree of hybridization between said oligonucleotide and its complement.

52. The composition of claim 51, wherein each said sequence has between ten and fifty bases.

53. The composition of claim 52, wherein each said sequence is between sixteen and forty bases in length, or between sixteen and thirty-five bases in length, or between eighteen and thirty bases in length, or between twenty and twenty-eight bases in length, or between twenty-one and twenty-seven bases in length, or between twenty-two and twenty-six bases in length.

54. The composition of claim 51, wherein each said sequence is of the same length as every other said sequence.

55. The composition of claim 54, wherein each said sequence is twenty-four bases in length.

56. The composition of claim 51, wherein no said sequence contains more than four contiguous bases that are identical to each other.

57. The composition of claim 51 wherein the number of G'"'"'s in each said sequence does not exceed L/4 where L is the number of bases in said sequence.

58. The composition of claim 57, wherein the number of G'"'"'s in each said sequence does not vary from the average number of G'"'"'s in all of the sequences of the set by more than one.

59. The composition of claim 58, wherein the number of G'"'"'s in each said sequence is the same as every other sequence of the set.

60. The composition of claim 58, wherein each said sequence is twenty-four bases in length and each said sequence contains 6 G'"'"'s.

61. The composition of claim 51, wherein, for each said sequence, there is at most six bases other than G between every pair of neighboring pairs of G'"'"'s.

62. The composition of claim 51, wherein at the 5′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

63. The composition of claim 51, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

64. The composition of claim 62, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a G.

65. The composition of claim 51, wherein under a said defined set of conditions, the maximum degree of hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 15%, more preferably 10%, more preferably 6%.

66. The composition of claim 65, wherein said set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C., and the oligonucleotides are covalently linked to microparticles.

67. The composition of claim 66, wherein under said defined set of conditions, the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5.

68. The composition of claim 51, comprising one hundred and sixty said molecules, or comprising one hundred and seventy said molecules, or comprising one hundred and eighty said molecules, or comprising one hundred and ninety said molecules, or comprising two hundred said molecules, or comprising two hundred and twenty said molecules, or comprising two hundred and forty said molecules, or comprising two hundred and sixty said molecules, or comprising two hundred and eighty said molecules, or comprising three hundred said molecules, or comprising four hundred said molecules, or comprising five hundred said molecules, or comprising six hundred said molecules, or comprising seven hundred said molecules, or comprising eight hundred said molecules, or comprising nine hundred said molecules, or comprising one thousand said molecules.

69. A composition of claim 51, wherein each said molecule is linked to a solid phase support so as to be distinguishable from a mixture of other said molecules by hybridization to its complement.

70. The composition of claim 69, wherein each molecule is linked to a defined location on a said solid phase support, the defined location for each said molecule being different than the defined location for different other said molecules.

71. The composition of claim 70, wherein each said solid phase support is a microparticle and each said molecule is covalently to a different microparticle than each other different said molecule.

72. (canceled)

96. A composition comprising a set of molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

M1≦

19/24×

L1, M2≦

17/24×

L1, M3≦

21/24×

L1, M4≦

18/24×

L1, M5≦

20/24×

L1, where L1 is the length of the shortest sequence of the pair, where;

M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment of the pair of sequences;

M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of the pair of sequences of maximum score, wherein;

the score of an alignment is determined according to the equation (A×

m)−

(B×

mm)−

(C×

(og+eg))−

(D×

eg)), wherein;

for each of (i) to (iv);

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

97. A composition comprising a set of molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

M1≦

19, M2≦

17, M3≦

21, M4≦

18, and M5≦

20, where;

M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment of the pair of sequences;

M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of the pair of sequences of maximum score, wherein;

the score of a said alignment is determined according to the equation 3A−

B−

3C−

D, wherein;

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment.