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US 2015/0176068 A1Published application
Methods of isothermal amplification using blocked primers
AssigneeGreat Basin Scientific
InventorsBrian Hicke, Robert D. Jenison, Madeline West
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Abstract
Methods for rapidly detecting clinically relevant mutations in the infectious genome of an agent are disclosed. The methods include use of a novel target and temperature dependent RNase H mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a target sequence such as a sequence in the the rpoB gene.
Claims21
A method of isothermal, nucleic acid sequence amplification, comprising:
- (a) mixing a target sequence with a blocked primer which includes a flap comprising a single ribonucleotide at its 5′ end and a blocking modification at its 3′-end;
- (b) contacting the mixture of step (a) with a thermostable RNase H enzyme and thermostable helicase-dependent amplification reagents comprising buffer, DNA polymerase, helicase, and deoxynucleotide triphosphates;
- (c) heating the mixture of step (b).
14-16. (canceled)
Dependencies are inferred from the claim text.