Nanopore Sequencing - Fluorescence

Fluorescence

An effective technique to determine a DNA sequence has been developed using solid state nanopores and fluorescence. This fluorescence sequencing method converts each base into a characteristic representation of multiple nucleotides which bind to a fluorescent probe strand-forming dsDNA. With the two color system proposed, each base is identified by two separate fluorescences, and will therefore be converted into two specific sequences. Probes consist of a fluorophore and quencher at the start and end of each sequence, respectively. Each fluorophore will be extinguished by the quencher at the end of the preceding sequence. When the dsDNA is translocating through a solid state nanopore, the probe strand will be stripped off, and the upstream fluorophore will fluoresce.

This sequencing method has a capacity of 50-250 bases per second per pore, and a four color fluorophore system (each base could be converted to one sequence instead of two), will sequence over than 500 bases per second. Advantages of this method are based on the clear sequencing readout—using a camera instead of noisy current methods. However, the method does require sample preparation to convert each base into an expanded binary code before sequencing. Instead of one base being identified as it translocates through the pore, ~12 bases are required to find the sequence of one base.