Abstract
A single molecule, long read-length, real-time sequencing by- synthesis technology has been developed by building a sequencer directly on the surface of a ∼ 10 nm quantum dot nanocrystal. Fluorescence resonance energy-transfer technology (FRET) is utilized for DNA sequence detection, in which signals from the quantum-dot labeled DNA polymerase plus 4 DNA-base-specific acceptor dyes are simultaneously detected. Precisely engineered sequencing-grade QdotTM nanocrystals are smaller than current commercially available materials (to increase FRET signals), and have an exctinction coefficient ∼100X greater than organic-dyes, allowing for very low levels of excitation power to be used while sequencing, acting as the FRET donor, the QdotTM-polymerase generates a correlated “photon-dip” for every inserted based (termed the “quantum-correlation-signal”), allowing for more accurate basecalling. Because the sequencer is not physically bound to any solid substrate, it can be exchanged (like a reagent) during midsequence runs, effectively replacing damaged non-functioning polymerases mid-reaction. Each exchange cycle lengthens the effective read-length of the sequencer. In this manner, the read-length can be continuously extended without “gaps”. Expanding upon this flexibility, after sequencing a particular length of DNA, the newly synthesized strand can be selectively removed. The original genomic DNA strand is then re-primed, QdotTM-polymerase sequencers are rebound, and the identical genomic DNA strand can be sequenced again, greatly increasing the net accuracy and not requiring circularization of genomic templates. In combining these features, the desired accuracy and read-length can be “tuned” by adjusting the number of reagent exchange cycles. Because each sequencing reaction can be completed in minutes, multiple exchange experiments can be performed per sequencing hour. These QdotTM-polymerase sequencers can also bind to ultra-long DNA segments (>10kb) at multiple positions along the length of the DNA and sequence while moving “horizontally” (parallel to TIRF field), enabling the possibility of “ordered-reads” for long-phased haplotype sequencing. Examples of real-time sequencing of homopolymeric, patterned, and complex templates will be shown.
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