TM leveling methods

• US 7,751,982 B2
• Filed: 09/26/2003
• Issued: 07/06/2010
• Est. Priority Date: 04/03/1998
• Status: Expired due to Fees

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Elitech Holding BV (ELITech Group SAS)
• Inventors: Gall, Alexander A., Dempcy, Robert O., Vermeulen, Nicolaas M. J., Lokhov, Sergey G., Kutyavin, Igor V., Afonina, Irina A.
• Primary Examiner(s): Brusca; John S
• Application Number:
  US10/672,429
• Publication Number:
  US 20050075491A1
• Time in Patent Office: 2,475 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Methods for designing an oligonucleotide sequence having a selected duplex stability are provided wherein the oligonucleotide portion comprises at least one modified base selected from universal bases, unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally having attached minor groove binders and reporter groups.

Claims

1. A method for designing a preferred oligonucleotide sequence having a selected duplex stability comprising:

a) providing an oligonucleotide sequence having N bases and N−

1 neighboring base pairs,

b) modifying the oligonucleotide sequence to provide modified oligonucleotide sequences wherein said modified oligonucleotide sequences comprise at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines;

c) calculating the duplex stabilities of said modified oligonucleotide sequences using an algorithm applying a nearest-neighbor model for duplex formation thermodynamics for each of the N−

1 neighboring base pairs, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases;

d) selecting a preferred oligonucleotide sequence from the modified oligonucleotide sequences having a selected duplex stability; and

e) creating an oligonucleotide comprising the preferred oligonucleotide sequence.

3. The method of any one of claim 1 or 2, wherein said oligonucleotide sequence is derived from a database source.

4. The method of claim 3, wherein said database source is GENBANK.

5. The method of any one of claim 1 or 2, wherein said at least one modified base is a member selected from the group consisting of a base attached to an amino acid, a polyamide nucleic acid (PNA), and a locked nucleic acid sugar.

6. The method of claim 5, wherein said modified base is attached to PNA.

7. The method of claim 5, wherein said modified base is attached to a locked nucleic acid sugar.

8. The method of any one of claim 1 or 2, wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.

9. The method of any one of claims 1 or 2, wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, and HOBuPPG.

10. The method of claim 1, wherein said oligonucleotide has attached to it one or more members selected from the group consisting of a minor groove binder, a fluorophore, and a quencher.

11. The method of claim 10, wherein said oligonucleotide has a minor groove binder attached thereto.

12. The method of claim 2 or 11, wherein said minor groove binder has a formula selected from the group consisting of:

13. The method of claim 11, wherein said minor groove binder is attached to the oligonucleotide via a quencher molecule.

14. The method of any one of claim 1 or 2, wherein said algorithm predicts the melting temperature (T_m) of said oligonucleotide with an accuracy of about +/−

2°

C.

15. The method of any one of claim 1 or 2, wherein said method is applied to establish appropriate conditions for hybridization, renaturation, mapping variations of base compositions of sequences, or determination of sequence complexity and divergence.

16. The method of any one of claim 1 or 2, wherein said oligonucleotide is a capture probe in an array.

17. The method of claim 6, wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.

18. The method of claim 7, wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.

19. The method of claim 1, wherein the oligonucleotide sequence comprises a universal base and wherein the modified oligonucleotide sequences comprise the universal base and at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines.

2. A method for designing a preferred oligonucleotide sequence having a selected duplex stability comprising:

a) providing an oligonucleotide sequence having N bases and N−

1 neighboring base pairs,

b) modifying the oligonucleotide sequence to provide modified oligonucleotide sequences wherein said modified oligonucleotide sequences comprises comprise at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines, and a minor groove binder;

c) calculating a melting temperature (T_m) of said modified oligonucleotide sequences using an algorithm applying nearest neighbor thermodynamic parameters for each of the N−

1 neighboring base pairs, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases;

d) selecting a preferred oligonucleotide sequence from the modified oligonucleotide sequences having the selected melting temperature; and

e) creating an oligonucleotide comprising the preferred oligonucleotide sequence.

20. The method of claim 2, wherein the oligonucleotide sequence comprises a universal base and wherein the modified oligonucleotide sequences comprise the universal base and at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines.