Composition and method for hot start nucleic acid amplification
View Patent ↗The present invention is directed to a new composition for performing a nucleic acid amplification reaction comprising (i) a thermostable DNA-Polymerase, (ii) a thermostable 3′-5′ Exonuclease, and (iii) at least one primer for nucleic acid amplification with a modified 3′ terminal residue which is not elongated by said thermostable DNA-Polymerase as well as methods for performing a PCR reaction using this composition. Furthermore, the method is directed to kits comprising such a composition.
1. A composition for nucleic acid amplification comprising:
a. a thermostable DNA polymerase,
b. a thermostable 3′-5′ exonuclease specific for double stranded DNA with a 5′ overhang and having no or reduced polymerase activity, and
c. at least one primer for nucleic acid amplification with a modified 3′ terminal residue which is not elongated by said thermostable DNA polymerase.
2. The composition of claim 1 wherein said is more active at temperatures between 37° C. and 72° C. and less active at temperatures below 37° C.
3. The composition of claim 1 wherein said thermostable exonuclease is either an exonuclease III homologue or a mutated DNA polymerase with no or reduced polymerase activity.
4. The composition of claim 1 further comprising a compound for detection of an amplification product.
5. The composition of claim 1 wherein at least one primer is modified at the 3′ end by a phosphate group or by a chemical moiety which is linked to the 3′ terminal deoxynucleotide via a phosphate group.
6. The composition of claim 1 wherein at least one primer has a 3′ terminal dideoxynucleotide residue.
7. A kit comprising a thermostable DNA polymerase, a thermostable 3′-5′ exonuclease specific for double stranded DNA with a 5′ overhang and having no or reduced polymerase activity, and at least one primer for nucleic acid amplification with a modified 3′ terminal residue which is not elongated by said thermostable DNA polymerase.
8. A method for amplifying a template nucleic acid comprising:
a. providing a reaction mixture comprising a thermostable DNA polymerase, a thermostable 3′-5′ exonuclease specific for double stranded DNA with a 5′ overhang and having no or reduced polymerase activity, and at least one primer which is modified at its 3′ terminal residue such that said primer is not elongated by the thermostable DNA polymerase and wherein the 3′ terminal residue of the primer is removed by the thermostable exonuclease,
b. removing said modification without addition of further reaction mixture components by raising the temperature of said reaction mixture, and
c. amplifying the template nucleic acid.