Methods and kits for 3'-end-tagging of RNA
AssigneeEpicentre Technologies Corporation
InventorsRamesh Vaidyanathan, Scott Kuersten, Ken Doyle
Life of this patent
Estimating the term…
Abstract
The present innovation provides methods and kits that enable rapid and efficient dual end-tagging of RNA to prepare libraries for analysis by applications such as next-generation RNA sequencing, qPCR, microarray analysis, or cloning. The methods do not require time-consuming and inefficient gel-purification steps that are common to methods known in the art. In addition, the present invention provides methods and kits for rapid, high-throughput enzymatic preparation of 5′-activated, 3′-blocked DNA oligonucleotides from standard, single-stranded DNA oligonucleotides.
Claims21
A method for enzymatic removal of excess activated donor DNA oligonucleotide from a sample following a bimolecular ligation reaction catalyzed by a single strand ligase enzyme comprising:
- (A) incubating the sample with TAP under conditions such that the excess 5′-App-DNA oligonucleotide is converted to 5′-phosphorylated donor DNA oligonucleotide;
- and
- (B) incubating the sample from step (A) with a DNA nuclease enzyme under conditions such that the 5′-phosphorylated donor DNA oligonucleotide is converted to deoxynucleotide monophosphates,
- wherein the incubating the sample with said TAP and DNA nuclease enzyme occurs in the same reaction vessel.
A method for enzymatic removal of excess activated donor DNA oligonucleotide from a sample following a bimolecular ligation reaction catalyzed by a single strand ligase enzyme comprising:
- (A) incubating the sample with TAP under conditions such that the excess 5-App-DNA oligonucleotide is converted to 5′-phosphorylated donor DNA oligonucleotide;
- and
- (B) incubating the sample from step (A) with a DNA nuclease enzyme under conditions such that the 5′-phosphorylated donor DNA oligonucleotide is converted to deoxynucleotide monophosphates,
- wherein steps (A) and (B) occur without intervening sample purification.
Dependencies are inferred from the claim text.