Journal
RNA
Volume 23, Issue 10, Pages 1582-1591Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.061184.117
Keywords
terminal deoxynucleotidyl transferase; dideoxy-UTP; oligonucleotide labeling; single-molecule FISH; RNA affinity purification
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Funding
- EMBL
- Deustche Forschungsgemeinschaft [DFG-FOR 2333]
- EMBL Interdisciplinary Postdoc Program (EIPOD) under Marie Curie COFUND actions
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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.
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