Carbodiimide Reaction with 5 Phosphates of DNA Phosphoramidate Formation

Conjugation via Carbodiimide Reaction with the 5 Phosphate of DNA (Phosphoramidate Formation) 

Carbodiimide modification of the phosphomonoester end groups on DNA molecules was first used in Khorana s lab to determine nucleotide sequences (Ralph et al., 1962). That early 

The formation of a phosphorimidazolide intermediate provides better reactivity toward amine nucleophiles than the EDC phosphodiester intermediate if EDC is used without added imidazole. The EDC phosphodiester intermediate also has a shorter half-life in aqueous conditions due to hydrolysis than the phosphorimidazolide. Although EDC alone will create nucleotide phosphoramidate conjugates with amine-containing molecules (Shabarova, 1988), the result of forming the secondary phosphorimidazolide-activated species is increased derivatiza-tion yield over carbodiimide-only reactions. 

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

Weigh out 1.25 mg of the carbodiimide EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodii mide hydrochloride Thermo Fisher) into a microfuge tube. 

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