Methods and kits for 3′-end-tagging of RNA
View Patent ↗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.
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.