Rapid detection of Dam methyltransferase activity based on the exonuclease III-assisted isothermal amplification cycle

Abstract

The DNA isothermal amplification cycle is an effective expansion strategy, which can overcome the inherent limitation of the target to signal ratio of 1 : 1. In this work, we fabricated an electrochemical biosensor for the rapid detection of Dam methyltransferase activity based on the exonuclease III (Exo III) assisted isothermal amplification cycle. First, double stranded DNA (ds-DNA) containing the symmetric sequence of 5′-GATC-3′ was immobilized on the magnetic bead surface. After the ds DNA was methylated by Dam MTase, it could be further cleaved by DpnI and formed a blunt end, which could lead to the digestion of a DNA strand by Exo III and release of a target single stranded DNA (ts-DNA). Then, the ts-DNA can further hybridize with DNA probes (DNA3) immobilized on the AuNPs/Au electrode surface and form a blunt end DNA–DNA duplex. Subsequently, the DNA3 in the DNA–DNA duplex can be digested by Exo III and release ts-DNA. The released ts-DNA can further hybridize with the unhybridized single stranded DNA (ss-DNA) and an isothermal amplification cycle is achieved. The method showed high sensitivity with a low detection limit of 0.031 unit per mL. Moreover, inhibition investigation indicates that Dam MTase activity can be inhibited by using 5-fluorouracil with an IC₅₀ value of 131.22 μM.