A heteroduplex-preferential Tm depressor for the specificity-enhanced DNA polymerase chain reactions

A macrocyclic tetraamine zinc(II) complex appended with two quinoline groups, Zn2+-1,7-bis(4-quinolylmethyl)-1,4,7,10- tetraazacyclododecane (Zn2+-Q2-cyclen), was successfully used as a novel additive to suppress nonspecific products in DNA polymerase chain reaction (PCR). In the presence of Zn2+-Q2-cyclen, the Tm drop of 20-bp heteroduplexes containing a noncomplementary basepair was greater than that of the corresponding homoduplex (i.e., primer DNA). Here, we applied such preferential DNA melting to a specificity-enhanced PCR using micromolar concentrations of Zn2+-Q2-cyclen. We demonstrated the selective amplification of target DNA fragments (i.e., the human heart sodium channel Nav1.5 gene) from genomic DNA or a cDNA library. The optimum condition for the specificity-enhanced PCR could be determined in the concentration range of 1-50 μM of Zn2+-Q2-cyclen.