Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof

• US 8,624,014 B2
• Filed: 12/21/2009
• Issued: 01/07/2014
• Est. Priority Date: 01/25/2001
• Status: Expired due to Term

• Current Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Original Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Inventors: Kobler, Daniel, Fieldhouse, Daniel
• Primary Examiner(s): NEGIN, RUSSELL SCOTT
• Application Number:
  US12/643,570
• Publication Number:
  US 20100311957A1
• Time in Patent Office: 1,478 Days
• Field of Search: None
• US Class Current: 536/24.2
• 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 oligonucleotides, each oligonucleotide in the composition either being free of cytosine residues and comprising guanosine residues or being free of guanosine residues and comprising cytosine residues,

wherein for each oligonucleotide that comprises cytosine residues, the cytosine residues are separated by between one and six non-cytosine residues, and for each oligonucleotide that comprises guanosine residues, the guanosine residues are separated by between one and six non-guanosine residues,

wherein the number of cytosine and guanosine residues present in each oligonucleotide in the composition does not exceed L/4 where L is the number of bases in the oligonucleotide,

wherein the length of each oligonucleotide in the composition differs by no more than five bases from the average length of all oligonucleotides in the composition,

wherein each oligonucleotide in the composition contains no more than 4 contiguous identical nucleotides,

wherein the number of guanosine and cytosine residues present in each oligonucleotide in the composition does not vary from the average number of guanosine and cytosine residues present in all other oligonucleotides of the composition by more than one, and

when each oligonucleotide of the composition is exposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C., the degree of cross-hybridization between the oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed 30% of the degree of hybridization between the oligonucleotide and an oligonucleotide fully complementary thereto.

2. The composition of claim 1, further characterized in that, each oligonucleotide in the composition contains either a cytosine or guanosine residue located within seven residues of an end of the oligonucleotide.

3. The composition of claim 1, wherein the length of each oligonucleotide in the composition is identical.

4. The composition of claim 3, wherein the number of guanosine and cytosine residues present in each oligonucleotide is the same.

5. The composition of claim 1, wherein the oligonucleotides are attached to a solid phase support.

6. The composition of claim 5, wherein the support is a planar substrate comprising a plurality of spatially addressable regions.

7. The composition of claim 1, wherein the oligonucleotides are covalently linked to microparticles.

8. The composition of claim 7, wherein the microparticles are spectrophotometrically unique and each unique microparticle has a different oligonucleotide attached thereto.

9. The composition of claim 1, wherein the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed 10% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide.

10. The composition of claim 1, wherein each oligonucleotide of the composition is at least ten nucleotides in length.

11. A composition comprising oligonucleotides, each oligonucleotide of the composition consisting of a sequence of ten to fifty nucleotide bases in length and for which, under a single set of hybridization conditions, the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 20% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide, and wherein each sequence is free of cytosine residues and comprises guanosine residues and, for each sequence, the number of guanosine residues does not exceed L/4 where L is the number of bases in said sequence.

12. The composition of claim 11, wherein each said sequence is of the same length as every other said sequence.

13. The composition of claim 12, wherein each said sequence is twenty-four bases in length.

14. The composition of claim 11, wherein the number of guanosine residues in each said sequence does not vary from the average number of guanosine residues in all of the sequences of the set by more than one.

15. The composition of claim 14, wherein each said sequence is twenty-four bases in length and each said sequence contains six guanosine residues.

16. The composition of claim 11, 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 guanosine residue.

17. The composition of claim 11, 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 guanosine residue.

18. The composition of claim 16, 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 guanosine residue.

19. The composition of claim 11, wherein each said oligonucleotide is linked to a solid phase support so as to be distinguishable from a mixture of other said oligonucleotides by hybridization to its complement.

20. The composition of claim 19, wherein each oligonucleotide is linked to a location on a said solid phase support that is different than the location for each other different said oligonucleotide.

21. The composition of claim 20, wherein each said solid phase support is a microparticle and each said oligonucleotide is covalently linked to a different microparticle than each other different said molecule oligonucleotide.

22. The composition comprising oligonucleotides, each oligonucleotide of the composition consisting of a sequence of ten to fifty nucleotide bases in length and for which, under a single set of hybridization conditions, the degree of cross-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 a complement to said oligonucleotide, and wherein each sequence is free of guanosine residues and comprises cytosine residues and, for each sequence, the number of cytosine residues does not exceed L/4 where L is the number of bases in said sequence.

23. The composition of claim 22, wherein each said sequence is of the same length as every other said sequence.

24. The composition of claim 23, wherein each said sequence is twenty-four bases in length.

25. The composition of claim 22, wherein the number of cytosine residues in each said sequence does not vary from the average number of cytosine residues in all of the sequences of the set by more than one.

26. The composition of claim 25, wherein each said sequence is twenty-four bases in length and each said sequence contains six cytosine residues.

27. The composition of claim 22, 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 cytosine residue.

28. The composition of claim 22, 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 cytosine residue.

29. The composition of claim 27, 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 cytosine residue.

30. A composition of claim 22, wherein each said oligonucleotide is linked to a solid phase support so as to be distinguishable from a mixture of other said oligonucleotides by hybridization to its complement.

31. The composition of claim 30, wherein each oligonucleotide is linked to a location on a said solid phase support that is different than the location for each other different said oligonucleotide.

32. The composition of claim 31, wherein each said solid phase support is a microparticle and each said oligonucleotide is covalently linked to a different microparticle than each other different said oligonucleotide.

33. A composition comprising oligonucleotides each attached to a solid support, each attached oligonucleotide in the composition either being free of cytosine residues and comprising guanosine residues or being free of guanosine residues and comprising cytosine residues,

wherein for each attached oligonucleotide that comprises cytosine residues, the cytosine residues are separated by between one and six non-cytosine residues, and for each attached oligonucleotide that comprises guanosine residues, the guanosine residues are separated by between one and six non-guanosine residues,

wherein the number of cytosine and guanosine residues present in each attached oligonucleotide in the composition does not exceed L/4 where L is the number of bases in the oligonucleotide,

wherein the length of each attached oligonucleotide in the composition differs by no more than five bases from the average length of all attached oligonucleotides in the composition,

wherein each attached oligonucleotide in the composition contains no more than 4 contiguous identical nucleotides,

wherein the number of guanosine and cytosine residues present in each attached oligonucleotide in the composition does not vary from the average number of guanosine and cytosine residues present in all other attached nucleotides of the composition by more than one, and

when each attached oligonucleotide of the composition is exposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C., the degree of cross-hybridization between the attached oligonucleotide and an oligonucleotide of the composition that is not fully complementary to the attached oligonucleotide does not exceed 30% of the degree of hybridization between the attached oligonucleotide and a fully complementary oligonucleotide to said attached oligonucleotide.

34. The composition of claim 33, further characterized in that, each attached oligonucleotide in the composition contains either a cytosine or guanosine residue located within seven residues of an end of the oligonucleotide.

35. The composition of claim 33, wherein the length of each attached oligonucleotide in the composition is identical.

36. The composition of claim 35, wherein the number of guanosine and cytosine residues present in each attached oligonucleotide is the same.

37. The composition of claim 33, wherein the support is a planar substrate comprising a plurality of spatially addressable regions.

38. The composition of claim 33, wherein the attached oligonucleotides are covalently linked to microparticles.

39. The composition of claim 38, wherein the microparticles are spectrophotometrically unique and each unique microparticle has a different oligonucleotide attached thereto.

40. The composition of claim 33, wherein the degree of cross-hybridization between a said attached oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed 10% of the degree of hybridization between said attached oligonucleotide and a complement to said attached oligonucleotide.

41. The composition of claim 33, wherein each attached oligonucleotide of the composition is at least ten nucleotides in length.