Nanopore sequencing

Nanopore sequencing is a third generation approach used in the sequencing of biopolymers- specifically, polynucleotides in the form of DNA or RNA. Nanopore sequencing is a third generation approach used in the sequencing of biopolymers- specifically, polynucleotides in the form of DNA or RNA. Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. At least one of these aforementioned steps is necessary in the procedure of any previously developed sequencing approach. Nanopore sequencing has the potential to offer relatively low-cost genotyping, high mobility for testing, and rapid processing of samples with the ability to display results in real-time. Publications on the method outline its use in rapid identification of viral pathogens, monitoring ebola, environmental monitoring, food safety monitoring, human genome sequencing, plant genome sequencing, monitoring of antibiotic resistance, haplotyping and other applications. Nanopore sequencing uses electrophoresis to transport an unknown sample through an orifice of 10−9 meters in diameter. A nanopore system always contains an electrolytic solution- when a constant electric field is applied, an electric current can be observed in the system. The magnitude of the electric current density across a nanopore surface depends on the nanopore's dimensions and the composition of DNA or RNA that is occupying the nanopore. Sequencing is made possible because, when close enough to nanopores, samples cause characteristic changes in electric current density across nanopore surfaces. The total charge flowing through a nanopore channel is equal to the surface integral of electric current density flux across the nanopore unit normal surfaces between times t1 and t2. Biological nanopore sequencing relies on the use of transmembrane proteins, called porins, that are embedded in lipid membranes so as to create size dependent porous surfaces- with nanometer scale 'holes' distributed across the membranes. Sufficiently low translocation velocity can be attained through the incorporation of various proteins that facilitate the movement of DNA or RNA through the pores of the lipid membranes. Alpha hemolysin (αHL), a nanopore from bacteria that causes lysis of red blood cells, has been studied for over 15 years. To this point, studies have shown that all four bases can be identified using ionic current measured across the αHL pore. The structure of αHL is advantageous to identify specific bases moving through the pore. The αHL pore is ~10 nm long, with two distinct 5 nm sections. The upper section consists of a larger, vestibule-like structure and the lower section consists of three possible recognition sites (R1, R2, R3), and is able to discriminate between each base. Sequencing using αHL has been developed through basic study and structural mutations, moving towards the sequencing of very long reads. Protein mutation of αHL has improved the detection abilities of the pore. The next proposed step is to bind an exonuclease onto the αHL pore. The enzyme would periodically cleave single bases, enabling the pore to identify successive bases. Coupling an exonuclease to the biological pore would slow the translocation of the DNA through the pore, and increase the accuracy of data acquisition. Notably, theorists have shown that sequencing via exonuclease enzymes as described here is not feasible. This is mainly due to diffusion related effects imposing a limit on the capture probability of each nucleotide as it is cleaved. This results in a significant probability that a nucleotide is either not captured before it diffuses into the bulk or captured out of order, and therefore is not properly sequenced by the nanopore, leading to insertion and deletion errors. Therefore, major changes are needed to this method before it can be considered a viable strategy. A recent study has pointed to the ability of αHL to detect nucleotides at two separate sites in the lower half of the pore. The R1 and R2 sites enable each base to be monitored twice as it moves through the pore, creating 16 different measurable ionic current values instead of 4. This method improves upon the single read through the nanopore by doubling the sites that the sequence is read per nanopore. Mycobacterium smegmatis porin A (MspA) is the second biological nanopore currently being investigated for DNA sequencing. The MspA pore has been identified as a potential improvement over αHL due to a more favorable structure. The pore is described as a goblet with a thick rim and a diameter of 1.2 nm at the bottom of the pore. A natural MspA, while favorable for DNA sequencing because of shape and diameter, has a negative core that prohibited single stranded DNA(ssDNA) translocation. The natural nanopore was modified to improve translocation by replacing three negatively charged aspartic acids with neutral asparagines.