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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.
1. 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 within about 5° C. of each other.
2. The composition of claim 1 , wherein said first oligonucleotide is within about ±2 bases of said second oligonucleotide.
3. The composition of claim 1 , 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.
4. The composition of claim 1 , 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.
5. The composition of claim 1 , 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, (NH 2 ) 2 PPPA, (NH 2 ) 2 PPPAOH, (NH 2 ) 2 BuPPAOH, (NH 2 ) 2 PPAI, and HOBuPPG.
6. The composition of claim 1 , wherein said second sequence has at least one modified base.
7. The composition of claim 6 , 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.
8. The composition of claim 6 , 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, (NH 2 ) 2 PPPA, (NH 2 ) 2 PPPAOH, (NH 2 ) 2 BuPPAOH, (NH 2 ) 2 PPAI, and HOBuPPG.
9. The composition of claim 1 , wherein said first sequence has a MGB attached thereto.
10. The composition of claim 1 , wherein said second sequence has a MGB attached thereto.
11. The composition of claim 1 , wherein said at least one modified base in said first sequence decreases stability of said first oligonucleotide.
12. The composition of claim 1 , wherein said at least one modified base in said first sequence increases stability of said first oligonucleotide.
13. The composition of claim 1 , wherein said first oligonucleotide and said second oligonucleotide are arrayed on a surface.
14. The composition of claim 13 , 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.
15. The composition of claim 13 , 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.
16. The composition of claim 13 , wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, each having T m s within about 2° C. of each other and basepair lengths within about 2 bases of each other.