A nanopore-based sequencing platform that can control the passage of DNA and accurately distinguish between all four bases has been developed by researchers at the University of Washington and University of Alabama at Birmingham. They claim their achievement effectively overcomes both these two longstanding hurdles to the development of an accurate nanopore sequencing system.
The platform developed by Washington’s Jens H. Gundlach, Ph.D., and colleagues, is based on an engineered form of the protein pore Mycobacterium smegmatis porin A (MspA), which is about 1.2 nm wide and 0.6 nm long. The MspA pore is incorporated into a lipid bilayer membrane bathed in potassium chloride, to which a voltage is applied to create an ion current flowing through the pore. Molecules translocating through the pore essentially generate a momentary, detectable change in current.
What makes this system so sensitive, however, is the integration of bacteriophage DNA polymerase (DNAP) phi29 into the pore, which effectively acts as a motor to pull the single-stranded template through the aperture one nucleotide at a time. While previous nanopore-based sequencing systems haven’t been capable of accurately discriminating between the four different nucleotides, the MspA-phi29 DNAP platform slows down translocation through the pore just enough to allow the telltale signal generated by each of different nucleotides to be identified. “The motor pulls the strand through the pore at a manageable speed of tens of milliseconds per nucleotide, which is slow enough to be able to read the current signal,” Dr. Gundlach explains.
The investigators report their achievement and experiments to confirm the system’s sensitivity in Nature Biotechnology in a paper titled “Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.”