Helicase-dependent amplification of RNA
View Patent ↗Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.
1. A method for helicase-dependent amplification of an RNA, comprising:
(a) adding to the RNA, a mixture comprising a reverse transcriptase, a helicase and a single strand binding protein unless the helicase is a thermostable helicase in which case the single strand binding protein is not required, a plurality of oligonucleotide primers and one or more polymerases; and (b) in the mixture, reverse transcribing the RNA to form a cDNA and amplifying, by helicase-dependent amplification, the cDNA wherein the amplification does not occur in the absence of the helicase as determined by gel electrophoresis.
2. A method according to claim 1 , wherein the helicase preparation comprises a UVrD helicase or homolog thereof.
3. A method according to claim 2 , wherein the UVrD helicase is a thermostable helicase.
4. A method according to claim 1 , wherein the helicase-dependent amplification is isothermal.
5. A method according to claim 4 , wherein the isothermal amplification occurs at a temperature in the range of 20° C.-75° C.
6. A method according to claim 1 , wherein the polymerase is Bst polymerase.
7. A method according to claim 1 , wherein the mixture further comprises an accessory protein.