A heteroduplex-preferential Tm depressor for the specificity-enhanced DNA polymerase chain reactions.
A macrocyclic tetraamine zinc(II) complex appended with two quinoline groups, Zn(2+)-1,7-bis(4-quinolylmethyl)-1,4,7,10- tetraazacyclododecane (Zn(2+)-Q2-cyclen), was successfully used as a novel additive to suppress nonspecific products in DNA polymerase chain reaction (PCR). In the presence of Zn(2+)-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 Zn(2+)-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 1-50muM of Zn(2+)-Q2-cyclen.
Kinoshita
Eiji
E
Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. kinoeiji@hiroshima-u.ac.jp
Kinoshita-Kikuta
Emiko
E
Koike
Tohru
T
D000818:Animals; D043822:Cyclodecanes; D006801:Humans; D009206:Myocardium / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods; D044367:Phase Transition; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D051381:Rats; D013696:Temperature
D043822:Cyclodecanes