Paper BI-ThP4
In Situ Surface Initiated Enzymatic DNA Polymerization: Potential for Multiplexed Molecular Detection

Thursday, November 3, 2011, 6:00 pm, Room East Exhibit Hall

We present a new technique called surface initiated enzymatic polymerization (SIEP), which uses 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 polymerize a long DNA chain while incorporating non-natural chemically reactive dNTPs to create new functional materials and to generate signal amplification. Previously, we have shown that fluorescent-dNTP can be directly polymerized by TdT. In this work, we will show the incorporation of amine- and aldehyde-modified dNTPs to impart reactive moieties into the polymerized DNA chain. We quantified the number of reactive dNTPs and their effect on the polymerization efficiency. We then further investigated the reactivity of the functional group for subsequent reactions, which include fluorescent dye conjugation for signal amplification, selective DNA metallization, and oligonucleotide conjugation to create a branched structure. We found that multiple reactive groups can be incorporated and they are active for subsequent reactions. For characterization of SIEP, we utilized a quartz crystal microbalance with dissipation monitoring (QCM-D) to monitor increase in surface mass during DNA polymerization in real-time. The mass increase versus concentrations of dNTP allows us to determine the reaction constant, which reflects the growth kinetics of DNA polymerization on the surface. In addition, we examined the effect of grafting density on the polymerization reaction and the conformation of DNA brushes.