Methods of isothermal amplification using blocked primers

摘要

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 rpoB gene.

主权项

1. A system for diagnostic testing, comprising:
a blocked primer which includes a flap comprising a single ribonucleotide at its 5′end and a blocking modification at its 3′ end; thermostable RNase H enzyme; and thermostable helicase-dependent amplification reagents, comprising: buffer, DNA polymerase, helicase, and deoxynucleotide triphosphates.