Analysis of DNA and single-base mutations using magnetic particles for purification, amplification and DNAzyme detection

• Published in: Analyst (London) Vol. 133; no. 7; p. 923
• Main Authors: Willner, Itamar, Cheglakov, Zoya, Weizmann, Yossi, Sharon, Etteri
• Format: Journal Article
• Language: English
• Published: England 01-01-2008
• Subjects: Animals; DNA - analysis; DNA, Catalytic - analysis; Humans; Luminescent Measurements; Magnetics; Point Mutation
• ISSN: 1364-5528
• DOI: 10.1039/b802015a

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.