Tm leveling compositions

• US 20060003349A1
• Filed: 04/19/2005
• Published: 01/05/2006
• Est. Priority Date: 04/03/1998
• Status: Active Grant

• Current Assignee: ELITechGroup Inc. (Bruker Corporation)
• 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 7,368,549 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 composition having a plurality of oligonucleotides, said composition comprising:

a first oligonucleotide with a first sequence having at least one modified base and a first T_m; and

a second oligonucleotide with a second sequence and a second T_m, wherein said first T_m and said second T_m are level.

111. The composition of claim 110, wherein said first T_m is within about 5°

C. of said second T_m.

112. The composition of claim 110, wherein said first oligonucleotide is within about ±

2 bases of said second oligonucleotide.

113. The composition of claim 110, wherein said at least one modified base in said first sequence is a member selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.

114. The composition of claim 110, wherein said at least one modified base in said first sequence is selected from the group consisting of 3-substituted pyrazolo[3,4-d]pryimidine and 5-substituted pyrimidine.

115. The composition of claim 110, wherein said at least one modified base in said first sequence 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.

116. The composition of claim 110, wherein said second sequence has at least one modified base.

117. The composition of claim 116, wherein said at least one modified base in said second sequence is a member selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.

118. The composition of claim 116, wherein said at least one modified base in said second sequence 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.

119. The composition of claim 110, wherein said first sequence has a MGB attached thereto.

120. The composition of claim 110, wherein said second sequence has a MGB attached thereto.

121. The composition of claim 110, wherein said at least one modified base in said first sequence decreases stability of said first oligonucleotide.

122. The composition of claim 110, wherein said at least one modified base in said first sequence increases stability of said first oligonucleotide.

123. The composition of claim 110, wherein said first oligonucleotide and said second oligonucleotide are arrayed on a surface.

124. The composition of claim 123, wherein said array comprises a solid support and a plurality of attached oligonucleotides, and wherein said plurality of oligonucleotides contain 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.

125. The composition of claim 123, wherein said array comprises a solid support and a plurality of attached oligonucleotides, and wherein said plurality of oligonucleotides contain at least one modified base selected from each of the groups consisting of 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines.

126. The composition of claim 123, wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, each having T_ms within about 2°

C. of each other and basepair lengths within about 2 bases of each other.