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Oligos modified with 5-OH me dC (5-hmc) are totally resistant to cleavage by Hpa II restriction enzyme. Msp I and Msp JI restriction enzymes will digest these oligos to almost completion. Usually there are 25-30% of resistant species remaining due to resistant protecting groups leftover during synthesis. Higher quantities of enzyme and longer incubation times (18-20 hrs) tends to increase digestion to greater than 90%. Oligos containing 5-hmc can be glucosylated by using T4 β-glucosyltransferase and thus resistant to Msp I digestion to discern between 5-mc and 5-hmc. The 5-OH group of 5-hmc is glucosylated and becomes completely resistant to Msp I digestion.
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