Analysis of DNA and single-base mutations using magnetic particles for purification, amplification and DNAzyme detection.
The amplified detection of DNA or of single-base mismatches in DNA is achieved by the use of nucleic acid-functionalized magnetic particles that separate the recognition duplexes and, upon amplification, yield chemiluminescence-generating DNAzymes as reporter units. The analysis of M13 phage ssDNA is achieved by the hybridization of the analyte to capture nucleic acid-functionalized magnetic particles followed by the binding of a DNA machine unit to the analyte domain. The magnetic separation of the multi-component-functionalized magnetic particles, followed by their reaction with polymerase, dNTPs, and the nicking enzyme (Nb.BbvCI) activate the autonomous synthesis of the horseradish peroxidase-mimicking DNAzyme that acts as chemiluminescent reporter. The single-base mutation in DNA is achieved by coupling of the DNA machine to the mutant DNA/capture nucleic acid-functionalized magnetic particles hybrid structure. The activation of the polymerization/nicking cycles yield the chemiluminescent reporting DNAzyme. The magnetic separation of the DNA recognition hybrids improves the signal-to-noise ratio of the analytical protocol as compared to related DNAzyme synthesizing schemes.
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. willnea@vms.huji.ac.il
Itamar
I
Willner
Zoya
Z
Cheglakov
Yossi
Y
Weizmann
Etteri
E
Sharon
D000818:Animals; D004247:DNA / Q000032:analysis*; D021881:DNA, Catalytic / Q000032:analysis*; D006801:Humans; D008163:Luminescent Measurements; D008280:Magnetics; D017354:Point Mutation
D021881:DNA, Catalytic; D004247:DNA