Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)
Figure 4
Synthesis and ligation of high efficiency adenylated cloning linkers. (a) An adenosine -phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its end and a free, reactive phosphate group on its end. (b) The synthetic, preactivated linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the end of the linker.
