Amplified On-Chip Fluorescent and Colorimetric Detection of DNA and RNA Hybridization by Surface-Initiated Enzymatic Polymerization

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We present a new on-chip, isothermal signal amplification technique for direct detection of unmodified DNA and RNA on microarrays using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3’-OH group of a DNA primer and yeast poly(A) polymerase (PaP), an RNA polymerase that catalyzes polyadenylation at the 3’-OH a RNA primer. We utilized their ability to catalyze the formation of long polynucleotide and their ability to incorporate unnatural nucleotide substrates such as fluorescent nucleotides into a long polymer chain of single stranded DNA. This methodology has several attractive attributes: (1) it can be carried out in situ, i.e., is on-chip; (2) it ensures that only the analytes of interest (targets) are labeled by introducing a detection label on the target after hybridization, so as to minimize false positive signals and background signal due to non-specific adsorption; (3) it provides signal amplification by incorporating multiple chromophores, or other labels per binding event, so that the output can be read by low-cost optical scanners or cell phone cameras; and (4) it can be carried out under isothermal conditions to minimize its technological complexity and make it field portable for point-of-care analysis.

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Journal: TechConnect Briefs
Volume: 3, Nanotechnology 2013: Bio Sensors, Instruments, Medical, Environment and Energy (Volume 3)
Published: May 12, 2013
Pages: 32 - 36
Industry sector: Medical & Biotech
Topics: Diagnostics & Bioimaging, Sensors - Chemical, Physical & Bio
ISBN: 978-1-4822-0586-2