Highly labeled DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates (dNTPs) were developed as a source of dNTPs for DNA polymerase. The particles were prepared by strand-displacement polymerization from a self-complementary circular template. Imaged by atomic force microscopy, these functionalized particles appear as condensed fuzzy balls with diameters between 50-150 nm. They emit a bright fluorescent signal, detected in 2 msec exposures with a signal-to-noise of 25 when imaged using a TIR fluorescence microscope. Keywordssingle molecule sequencing; phosphate-labeled nucleotide; DNA nanoball; SYBR; fluorescent Next generation sequencing efforts are utilizing a variety of new technologies with the aim of reducing the cost of sequencing a human genome to $1000. [1][2][3][4] Sequencing by ligation, hybridization, denaturation, exonuclease, cyclic synthesis, and through nanopores are some of the emerging technologies that are evolving the way we acquire DNA sequences. Our own efforts towards producing a next generation sequencer focused on using single molecule DNA sequencing to produce a high-throughput, low cost sequencing methodology. As with other single molecule sequencing approaches, detecting and correctly identifying individual bases is a daunting challenge. To help overcome the limitations inherent in single molecule detection schemes, we developed a system that uses a highly labeled DNA nanoball for identifying incorporated nucleotides. The highly labeled DNA nanoballs provide small, highly-charged, yet extremely bright fluorescent sources from which to detect binding events with millisecond exposures and high signal-to-noise ratios. Similarly designed DNA nanostructures are also currently being used in other next-generation sequencing endeavors. 5 Using these bright DNA nanostructures, we investigated a modified form of sequencing by synthesis, in which it is necessary to detect the addition of a nucleotide as it is incorporated in real-time into the growing DNA strand.jon.anderson@licor.com. SUPPORTING INFORMATION PARAGRAPH Detailed experimental procedures covering the production and labeling of our DNA nanoballs is included in the supporting information section. These procedures include: Primed template for nanoparticle construction, Rolling circle amplification for Nanoparticle construction, Sucrose purification of DNA nanoparticle, dCTP-PEG8-amine, Amino acid bridge attachment to modified triphosphate, SYBR ® 101-SE conjugation with modified nucleotide triphosphate, Derivatization of amino-nanoparticle to thiol reactive nanoparticle, and Covalent attachment of nucleotide-dye conjugates to nanoparticle. We classify this method of single-molecule sequencing as Field-Switch Sequencing 6 , where a surface bound polymerase containing a primed single-stranded DNA template is provided with labeled nanoballs containing attached dNTP's. In this method the nanoballs would be moved near the polymerase using an electric field, allowing the binding and eventual incorporation of the ...