Enzymatic production of single-molecule FISH and RNA capture probes
- Corresponding author: imre.gaspar{at}embl.de
Abstract
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling—including conjugation, enzymatic synthesis, and product purification—can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
Keywords
- terminal deoxynucleotidyl transferase
- dideoxy-UTP
- oligonucleotide labeling
- single-molecule FISH
- RNA affinity purification
Footnotes
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.061184.117.
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Freely available online through the RNA Open Access option.
- Received February 19, 2017.
- Accepted June 22, 2017.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.