Preparation of short cytosine-modified oligonucleotides by nicking enzyme amplification reaction

A method for enzymatic production of short (10-20 nt) cytosine-modified oligonucleotides was developed by nicking enzyme amplification reaction using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease and 5-substituted dCTP derivatives. The methodology including isolation was scaled up to nanomolar amounts and was proved to be suitable for production of diverse base-modified short single-stranded oligonucleotides (inaccessible by other enzymatic methods) that are of potential interest as labelled primers or functionalized aptamers.