Systems and methods for predicting oligonucleotide melting temperature (TmS)

• US 20030235822A1
• Filed: 06/18/2002
• Published: 12/25/2003
• Est. Priority Date: 04/03/1998
• Status: Active Grant

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Vermeulen, Nicolaas M.J., Gall, Alexander A., Dempcy, Robert O., Lokhov, Sergey G., Kutyavin, Igor V., Singer, Michael J., Afonina, Irina A.
• Granted Patent: US 7,715,989 B2
• Time in Patent Office: Days
• US Class Current: 435/6.18
• 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Software systems and methods for predicting the melting temperature (T_m) and other characteristics of oligonucleotides, including modified oligonucleotides. Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Claims

1. A computer-implemented method of predicting the melting temperature (T_m) of an oligonucleotide sequence, comprising:

identifying an oligonucleotide having a sequence of N bases; and

calculating a melting temperature (T_m) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases.

2. The computer-implemented method of claim 1, wherein identifying includes receiving a polynucleotide sequence from Genbank.

3. The computer-implemented method of claim 1, wherein identifying includes receiving a user input polynucleotide sequence.

4. The computer-implemented method of claim 1, wherein identifying includes receiving a polynucleotide sequence and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.

5. The computer-implemented method of claim 1, wherein said identified sequence includes at least one modified base.

6. The computer-implemented method of claim 5, wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid, a locked nucleic acid sugar and a universal base.

7. The computer-implemented method of claim 5, wherein said at least one modified base includes a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, HOBUPPA, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, (NH2)₂PPABr, (NH₂)₂PPACI, PPAI, PPABr, PPACl and HOBuPPG.

8. The computer-implemented method of claim 5, wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.

9. The computer-implemented method of claim 8, wherein said MGB has a formula selected from the group consisting of:

10. The computer-implemented method of claim 1, further comprising:

retrieving said nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory.

24. The computer-implemented method of claim 5, wherein said identified oligonucleotide sequence has attached to it one or more of aminor groove binder (MGB), a fluorophore (Fl) and a quencher (Q).

25. The computer-implemented method of claim 9, where the minor groove binder is attached to the oligonucleotide via a quencher molecule.

11. A computer readable medium including computer code for controlling a processor to calculate the melting temperature (T_m) of an oligonucleotide, the computer code including instructions to:

identify an oligonucleotide sequence responsive to user input, said sequence having N bases;

retrieve nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases; and

calculate a melting temperature (T_m) of the identified oligonucleotide sequence using the retrieved nearest neighbor thermodynamic parameters.

12. The computer-readable medium of claim 11, wherein said user input includes a Genbank code, wherein said instructions to identify include instructions to automatically retrieve a polynucleotide sequence from Genbank over a network using said input Genbank code.

13. The computer-readable medium of claim 11, wherein said user input includes a polynucleotide sequence.

14. The computer-readable medium of claim 11, wherein said instructions to identify includes instructions to identify one of a portion of a received polynucleotide sequence and a sequence complementary to a portion of the received sequence.

15. The computer-readable medium of claim 11, wherein said identified sequence includes at least one modified base.

16. The computer-readable medium of claim 15, wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid, a locked nucleic acid sugar and a universal base.

17. The computer-readable medium of claim 15, wherein said at least one modified base includes a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, HOBUPPA, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, (NH₂)₂PPABr, (NH₂)₂PPACl, PPAI, PPABr, PPACl, and HOBuPPG.

18. The computer-readable medium of claim 15, wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.

19. The computer-readable medium of claim 18, wherein said MGB has a formula selected from the group consisting of:

20. A computer system for predicting the melting temperature (T_m) of an oligonucleotide sequence, comprising:

a memory unit for storing nearest neighbor thermodynamic parameters;

means for identifying a nucleotide sequence having N bases, wherein at least one of said N bases includes a modified base; and

processing means, coupled to the memory unit, for retrieving nearest neighbor parameters for each of the N−

1 neighboring base pairs in the identified sequence from the memory unit, and for calculating a melting temperature (T_m) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases.

21. The system of claim 20, wherein the means for identifying includes means for receiving a user entered nucleotide sequence.

22. The system of claim 20, wherein the means for identifying includes means for receiving a GenBank GI number specifying a nucleotide sequence stored at a GenBank server, the system further including means for retrieving the specified sequence from a GenBank server.

23. The system of claim 20, wherein the means for identifying includes means for receiving a polynucleotide sequence having N or greater bases and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.