Oligonucleotide inhibitor of DNA polymerases
The invention comprises a reversible oligonucleotide inhibitor of nucleic acid polymerases. Methods of designing said inhibitors and using said inhibitors in amplification and detection of nucleic acid, particularly detection of RNA by RT-PCR are also disclosed.
1. A reversible inhibitor of nucleic acid polymerases comprising a single-stranded DNA oligonucleotide having more than one regions of double-stranded secondary structure and comprising SEQ ID NO: 1, wherein more than one thymine bases are replaced with uracil bases only in at least one of the more than one regions of double-stranded secondary structure, and wherein the more than one regions of double-stranded secondary structure are stable under ambient temperature in a PCR mixture.
2. A kit for amplifying a target nucleic acid containing a reversible inhibitor of a nucleic acid polymerase comprising a single-stranded DNA oligonucleotide having more than one regions of double-stranded secondary structure and comprising SEQ ID NO: 1, wherein more than one thymine bases are replaced with uracil bases only in at least one of the more than one regions of double-stranded secondary structure, and wherein the more than one regions of double-stranded secondary structure are stable under ambient temperature in a PCR mixture.
3. The kit of claim 2 , further comprising a uracil-N-glycosylase.
4. A reaction mixture for amplifying a target nucleic containing a reversible inhibitor of a nucleic acid polymerase comprising a single-stranded DNA oligonucleotide having more than one regions of double-stranded secondary structure and comprising SEQ ID NO: 1, wherein more than one thymine bases are replaced with uracil bases only in at least one of the more than one regions of double-stranded secondary structure, and wherein the more than one regions of double-stranded secondary structure are stable under ambient temperature in a PCR mixture.
5. The reaction mixture of claim 4 , further comprising uracil-N-glycosylase.
6. The reaction mixture of claim 5 , further comprising spermine.
7. The reversible inhibitor of claim 1 comprising SEQ ID NO: 4.
8. The kit of claim 3 , further comprising a spermine.
9. The reversible inhibitor of claim 1 comprising SEQ ID NO: 2.
10. The reversible inhibitor of claim 1 comprising SEQ ID NO: 3.