METHODS AND KITS FOR 3'-END-TAGGING OF RNA

• US 20140179562A1
• Filed: 09/13/2013
• Published: 06/26/2014
• Est. Priority Date: 11/05/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Epicentre Technologies Corporation (Illumina Incorporated)
• Inventors: Vaidyanathan, Ramesh, Kuersten, Scott, Doyle, Ken
• Granted Patent: US 9,790,540 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

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.

Claims

8-1. The kit of claim 1, wherein the one or more RNA oligonucleotides are 5′

and 3′

hydroxyl RNA oligonucleotides comprising one of a next generation sequencing adaptor sequence, a RNA polymerase promoter sequence or a restriction endonuclease site.