End-labeling oligonucleotides with chemical tags after synthesis.
Many experimental strategies for determining nucleic acid function require labeling the nucleic acid with radioisotopes or a chemical tag. Labels enable nucleic acid detection, yield information about its state, and can serve as a handle by which the nucleic acid and associated factors can be purified from a mixture. Radioactive phosphate is commonly added to the 5' or 3' end of an oligonucleotide post synthesis using enzyme-catalyzed reactions. In contrast, chemical tags are usually added during synthesis or using reactive groups that are incorporated during synthesis. Here, we present protocols for post-synthetic conjugation of chemical tags to unmodified RNA or DNA oligonucleotides. The approach can be used to attach fluorescent dyes and biotin groups to oligonucleotides and to immobilize oligonucleotides to a solid support. Oligonucleotides tagged with fluorescent dyes are readily detected in both gel- and plate reader-based assays, while biotin- or resin-conjugated oligonucleotides are useful tools for affinity purification. Fluorescent end-labeling provides several advantages over radioactive labeling, reducing radioactivity-associated hazards and yielding a labeled molecule that does not decay while providing the sensitivity required for many procedures.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.
N Ruth
NR
Zearfoss
Sean P
SP
Ryder
D060326:Chemistry Techniques, Synthetic; D004247:DNA / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
D009841:Oligonucleotides; D012313:RNA; D004247:DNA