Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis.
In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 x 10(-11), 2.4 x 10(-11) and 3.0 x 10(-11)M, respectively. The multiplex assay also provided good specificity without any cross-reaction.
Li
Xuemei
X
Department of Chemistry, Nanjing University, Nanjing, China.
Zhan
Zhiming
Z
Zhang
Shusheng
S
Chen
Hongyuan
H
D001360:Avidin / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C083986:avidin-horseradish peroxidase complex; D001360:Avidin; D001710:Biotin; D004247:DNA; D006735:Horseradish Peroxidase