The mechanism underlying DNA charge transport is intriguing. However, poor conductivity of DNA makes it difficult to detect DNA charge transport. Metallic DNA (M-DNA) has better conducting properties than native DNA. Ni2+ may chelate in DNA and thus enhance DNA conductivity. On the basis of this finding, it is possible to reveal the mechanisms underlying DNA charge transport. The conductivity of various Ni-DNA species such as single-stranded, full complement, or mismatched sequence molecules was systematically tested with ultraviolet absorption and electrical or chemical methods. The results showed that the conductivity of single-stranded Ni-DNA (Ni-ssDNA) was similar to that of a native DNA duplex. Moreover, the resistance of Ni-DNA with a single basepair mismatch was significantly higher than that of fully complementary Ni-DNA duplexes. The resistance also increased exponentially as the number of mismatched basepairs increased linearly after the tunneling current behavior predicted by the Simmons model. In conclusion, the charges in Ni 2+-doped DNA are transported through the Ni2+-mediated π-π stacking corridor. Furthermore, Ni-DNA acts as a conducting wire and exhibits a tunneling barrier when basepair mismatches occur. This property may be useful in detecting single basepair mismatches.