MODIFIED RNASE H ENZYMES AND THEIR USES

摘要

The invention provides a provides improvements to assays that employ RNase H cleavage for biological applications related to nucleic acid amplification and detection, where the RNase H has been reversibly inactivated.

主权项

1. A method of copying a target RNA molecule in a sample to produce a cDNA, the method comprising the steps of:
a) treating said sample in a reaction mixture comprising a polymerase, deoxyribonucleoside triphosphates, a buffer, a RNase H enzyme that is reversibly inactive, a first primer, wherein the first primer is blocked at or near the 3′-end of the first primer with a blocking group that is removable with an RNase H enzyme, and wherein the first primer is complementary to the target RNA to hybridize and form a double-stranded product; b) raising the temperature to activate the RNase H enzyme; c) hybridizing the first primer with the target RNA at an appropriate temperature; d) treating the double-stranded product with RNase H to remove the blocking group; and e) polymerizing the first primer to form a cDNA complementary to the target RNA.