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

V. Tjong, H. Yu, A. Hucknall, A. Chilkoti
Duke University, US

Keywords: surface-initiated, polymerization, enzyme, terminal deoxynucleotidyl transferase, poly (A) polymerase, RNA, mRNA, miRNA, fluorescent nucleotides, signal amplification, incorporation, DNA microarray, gold nanoparticle, colorimetric


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.