Methods and kits for 3′-end-tagging of RNA

• US 9,790,540 B2
• Filed: 09/13/2013
• Issued: 10/17/2017
• 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
• Primary Examiner(s): Thomas, David
• Application Number:
  US14/026,239
• Publication Number:
  US 20140179562A1
• Time in Patent Office: 1,495 Days
• Field of Search: None
• 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

1. A kit for preparing a library of nucleic acid molecules from one or more RNA acceptor oligonucleotides comprising:

a) one or more 5′

-adenylated, 3′

-blocked DNA oligonucleotides;

b) a RNA ligase or RNA ligases;

c) a ligation buffer;

d) Tobacco Acid Pyrophosphatase (TAP) or Yeast 5′

-Deadenylase;

e) RecJ Exonuclease or Lambda Exonuclease; and

f) a reaction buffer.

2. The kit of claim 1, further comprising one or more first-strand cDNA synthesis primers;

a reverse transcriptase enzyme;

dNTPs; and

one or more PCR primers.

3. The kit of claim 1, wherein the nucleic acid molecules comprise RNA or cDNA.

4. The kit of claim 1, wherein the one or more 5′

adenylated, 3′

-blocked DNA oligonucleotides are DNA oligonucleotide donors that have a 5′

-adenylated nucleotide but do not have a 3′

-hydroxyl nucleotide (5′

-App-DNA-X).

5. The kit of claim 1, further comprising one or more 5-tagging RNA oligonucleotides, wherein the one or more 5′

-tagging RNA oligonucleotides are 5′

- and 3′

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

6. The kit of claim 1, wherein the RNA ligase or RNA ligases is selected from the group consisting of T4 RNA Ligase 1, T4 RNA Ligase 2, truncated T4 RNA Ligase 2, a mixture comprising T4 RNA ligase 1 and truncated T4 RNA ligase 2, and bacteriophage TS2126 ligase.

7. The kit of claim 6, wherein one of the RNA ligase or RNA ligases is truncated T4 RNA Ligase 2.

8. The kit of claim 1, wherein the exonuclease is Rec J exonuclease.

9. The kit of claim 1, further comprising a ribonuclease inhibitor.

10. The kit of claim 1, wherein the one or more 5′

-adenylated, 3′

-blocked DNA oligonucleotides comprise one or more of a next generation sequencing adaptor sequence, a RNA polymerase promoter sequence, and a restriction endonuclease site.

11. The kit of claim 5, wherein the one or more 5′

-tagging RNA oligonucleotides are 5′

- and 3′

-hydroxyl RNA oligonucleotides.

12. The kit of claim 1, further comprising one or more 5′

-tagging RNA oligonucleotides that are 5′

- and 3′

-hydroxyl RNA oligonucleotides, wherein the 5′

-adenylated, 3′

-blocked DNA oligonucleotides and 5′

-tagging RNA oligonucleotides each comprise at least one sequence independently selected from the group consisting of a next generation sequencing adaptor sequence, a RNA polymerase promoter sequence, and a restriction endonuclease site.

13. The kit of claim 1, wherein the one or more 5′

-adenylated, 3′

-blocked DNA oligonucleotides comprise a 3′

-blocking group that is a dideoxynucleotide.