AVS 56th International Symposium & Exhibition
    Biomaterial Interfaces Thursday Sessions
       Session BI-ThP

Paper BI-ThP12
Enzymatic DNA Polymerization: Potential as Signal Amplification

Thursday, November 12, 2009, 6:00 pm, Room Hall 3

Session: Biomaterial Interfaces Poster Session II (Arrays, Sensing, Micro/Nanofabrication, SPM)
Presenter: V. Tjong, Duke Univerisity
Authors: V. Tjong, Duke Univerisity
A. Hucknall, Duke Univesity
H. Yu, Duke University
A. Chilkoti, Duke University
Correspondent: Click to Email
We have developed a new technique for on-chip, isothermal signal amplification using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer. We utilized TdT's ability to incorporate non-natural fluorescent dNTPs into a long polymer chain of single stranded DNA (ssDNA). We quantified the TdT mediated signal amplification on the surface by immobilizing ssDNA oligomers on a glass surface followed by surface initiated enzymatic polymerization of DNA. We examined the effect of the concentration of the different natural dNTPs, and the molar ratio of fluorescent dNTPs to natural dNTPs on the length of the polymerized DNA strand and the degree of fluorophore incorporation. These experiments allowed us to optimize the polymerization conditions to incorporate a large number of fluorescent nucleotides (up to ~100 fluorescent dNTP/chain) into the ssDNA chain catalyzed by TdT. For Cy3-labeled dATP, this translated to a maximum of ~40 fold signal amplification through the incorporation of multiple fluorophores into the extended DNA chain. This methodology has the attractive attributes that it is both isothermal and on-chip –as the fluorophores are covalently incorporated into a ssDNA chain that is grown from a tethered DNA strand at 37 ºC. We anticipate the use of this amplification modality for the development of sandwich fluoro-immunoassays and DNA microarrays where binding of the detection Ab in a sandwich assay or the target strand in a DNA microarray provides the 3'-OH groups necessary to initiate on-chip fluorescence amplification of the binding event.