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RNA. 2017 Oct;23(10):1582-1591. doi: 10.1261/rna.061184.117. Epub 2017 Jul 11.

Enzymatic production of single-molecule FISH and RNA capture probes.

Author information

1
Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg 69117, Germany.

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:

RNA affinity purification; dideoxy-UTP; oligonucleotide labeling; single-molecule FISH; terminal deoxynucleotidyl transferase

PMID:
28698239
PMCID:
PMC5602115
DOI:
10.1261/rna.061184.117
[Indexed for MEDLINE]
Free PMC Article

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