Amplified Fluorescence Detection of DNA and RNA Hybridization by Surface Initiated Enzymatic Polymerization ( SIEP )

Detection of nucleic acid on-chip has been of great interest due to its potential as research tools and for diagnostic applications. We present a new on-chip, isothermal signal amplification technique for the 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 an RNA primer. We utilized their ability to catalyze the formation of long polynucleotide and TdT’s ability to incorporate unnatural nucleotide substrates such as fluorescent nucleotides (F-dNTP) into a long polymer chain of single stranded DNA (ssDNA) from the 3’-OH of a target DNA or RNA that is tethered on the surface through hybridization. We obtained dose reponse curves of hybridized DNA and RNA with LOD in ~1-10 pM.