TM leveling methods

• US 20050075491A1
• Filed: 09/26/2003
• Published: 04/07/2005
• 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., Afonina, Irina A.
• Granted Patent: US 7,751,982 B2
• Time in Patent Office: Days
• US Class Current: 536/23.100
• 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

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

1-109. -109. (canceled)

110. A thermodynamic method for predicting duplex stability of an oligonucleotide sequence comprising at least one modified nucleotide base, said method comprising:

a) providing an oligonucleotide having a sequence of N bases, wherein said oligonucleotide comprises at least one modified base; and

b) calculating duplex stability of said oligonucleotide 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.

112. The method of any one of claims 110 or 111, wherein said oligonucleotide sequence is derived from a database source.

113. The method of claim 112, wherein said oligonucleotide sequence is derived from Genbank.

114. The method of any one of claims 110 or 111, 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.

115. The method of claim 114, wherein said modified base is attached to PNA.

116. The method of claim 114, wherein said modified base a locked nucleic acid sugar.

117. The method of any one of claims 110, 111, 115 or 116, wherein said oligonucleotide comprising at least one modified base has superior mismatch discrimination, in comparison to unmodified nucleotides.

118. The method of any one of claims 110 or 111, wherein said at least one modified base is a member selected from the group consisting of a universal base, PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, and HOBuPPG.

119. The method of any one of claims 110 or 111, 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.

120. The method of claim 119, wherein said oligonucleotide sequence has a minor groove binder attached thereto.

121. The method of claim 120, wherein said minor groove binder has a formula selected from the group consisting of:

122. The method of claim 120, wherein said minor groove binder is attached to the oligonucleotide via a quencher molecule.

123. The method of any one of claims 110 or 111, wherein said algorithm predicts the melting temperature (T_m) of said oligonucleotide with an accuracy of about +/−

2°

C.

124. The method of any one of claims 110 or 111, wherein said method is applied in establishing appropriate conditions for hybridization, renaturation, mapping variations of base compositions of sequences or determination of sequence complexity and divergence.

125. The method of any one of claims 110 or 111, wherein said oligonucleotide is a capture probe in an array.

111. A method for predicting the melting temperature (T_m) of an oligonucleotide sequence comprising at least one modified nucleotide base, said method comprising:

a) providing an oligonucleotide having a sequence of N bases, wherein said oligonucleotide comprises at least one modified base; and

b) calculating a melting temperature (T_m) of said oligonucleotide 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.