Synthetic nucleic acids for polymerization reactions
View Patent ↗Compositions and methods are provided for inhibiting a polymerase from replicating non target DNA at a temperature below the amplification reaction temperature. The inhibitor is a synthetic nucleic acid which is single stranded but folds to form at least one double stranded region designed to melt at a temperature which is lower than the amplification reaction temperature, and at least one single stranded region where the single stranded region at the 5′ end contains at least one uracil or inosine and optionally a sequence at the 3′ end contains one or more derivative nucleotide or linkages.
1. An aqueous solution, comprising:
(a) a thermostable polymerase that is active at a temperature of at least 65° C.; and
(b) an oligonucleotide that has:
(i) a double-stranded region having a T m of less than 65° C.,
(ii) a 5′ overhang comprising at least one uracil or inosine, and
(iii) a modified nucleotide or linkage that makes the 3′ end non-extendible or resistant to nuclease activity,
wherein the oligonucleotide is capable of inhibiting the thermostable polymerase when the aqueous solution is at a temperature of below 37° C. but not at a temperature of 65° C. or greater.
2. The aqueous solution of claim 1 , wherein the polymerase is a thermostable bacterial polymerase.
3. The aqueous solution of claim 1 , wherein the polymerase is a thermostable archael polymerase.
4. The aqueous solution of claim 1 , wherein the polymerase is a variant of a wild type thermostable polymerase.
5. The aqueous solution of claim 1 , wherein the polymerase has an amino acid sequence that is at least 93% identical to SEQ ID NO:25.
6. The aqueous solution of claim 1 , wherein the polymerase has an amino acid sequence that is at least 93% identical to SEQ ID NO:25 and comprises at least amino acid substitution at an position corresponding to 278, 307, and/or 402 in SEQ ID NO:25.
7. The aqueous solution of claim 1 , wherein the oligonucleotide and the thermostable polymerase are present in the solution at a molar ratio of between 0.5:1 to 10:1.
8. The aqueous solution of claim 1 , further comprising dNTPs and primers.
9. The aqueous solution of claim 1 , wherein the double-stranded region is 4-40 nucleotides in length.
10. The aqueous solution of claim 1 , wherein the double-stranded region is 6-60 nucleotides in length.
11. The aqueous solution of claim 1 , wherein the uracil or inosine is positioned at the fourth position in the 5′ single-strand overhang, numbered from the 3′ end of single-stranded portion of 5′ overhang.
12. The aqueous solution of claim 1 , wherein the overhang comprises at least 2 uracils or inosines.
13. The aqueous solution of claim 1 , wherein the oligonucleotide comprises a modified nucleotide that makes the 3′ end not extendible.
14. The aqueous solution of claim 13 , wherein the oligonucleotide comprises dideoxynucleotide, inverted base or amino-modified nucleotide at the 3′ end.
15. The aqueous solution of claim 1 , wherein the oligonucleotide comprises a linkage or modified nucleotide that makes the 3′ end resistant to nuclease activity.
16. The aqueous solution of claim 15 , wherein the oligonucleotide comprises a phosphorothioate linkage at or near the 3′ end.