Compositions and methods for polynucleotide sequencing
A technology of polynucleotides and nucleotides, applied in biochemical equipment and methods, measuring devices, microbiological determination/inspection, etc., can solve problems such as poor sequencing accuracy
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Embodiment I
[0198] Step-by-step translocation steps using Hel308 helicase
[0199] Example 1 describes the stepwise translocation steps observed using an exemplary Hel308 helicase.
[0200] The lipid bilayer is composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti polar lipid). The bilayer spans a pore of ~20 microns in horizontal diameter in Teflon. M2-NNN-MspA was added to the grounded side of the bilayer at a concentration of -2.5 ng / ml. Once inserted into a single well, the compartment is flushed with assay buffer to avoid further insertion. An Axopatch-200B patch clamp amplifier (Axon Instruments) applied a voltage of 180 mV across the bilayer and measured the ionic current. The analog signal was low-pass filtered at 50 kHz using a 4-pole Bessel filter and then digitized at 5 times the low-pass filtered frequency. Data acquisition was controlled using conventional software (National Instruments) written in LabWindows / CVI. Compartments of ~60 μl on both sides of the bil...
Embodiment II
[0206] Relationship between ATP concentration and stepwise translocation steps
[0207] Example II describes the effect of ATP concentration on the residence time of the stepwise metathesis step.
[0208] To further elucidate the biochemical mechanism of the stepwise translocation step, the residence time of the stepwise translocation step was examined at different ATP concentrations. The cis and trans wells were first filled with a buffer solution consisting of 400 mM KCl, 10 mM HEPES, pH8. A lipid bilayer composed of DPhPC was formed by coating a mixture of hexadecane and fat on ~25 μm diameter Teflon pores, and conductometric measurements were performed to ensure a gigaohm seal between the lipid bilayer and Teflon pores ( Gigaohmseal). All electrical measurements were performed using an Axopatch 200B patch clamp amplifier connected to a pair of Ag / AgCl electrodes connected to the cis and trans wells. After membrane formation, MspA nanopores were injected into the cis por...
Embodiment III
[0214] Use of stepwise translocation steps in polynucleotide sequencing
[0215] Example III describes the improved sequencing accuracy by using two stepwise translocation steps from a complete translocation cycle compared to using a single signal from a complete translocation cycle.
[0216] Since the MspA "read-head" is sensitive to 4-nucleotide (4-mer) stretches within the constriction, the slave measurements correspond to all 4-mers seen in the MspA nanopore A tetramer map of the combined currents yields current traces. For further details on measuring currents corresponding to 4-mer combinations, see Laszlo et al., "Decoding long nanopore sequencing reads of natural DNA," Nature Biotechnology 32:829-833 (2014). However, it is understood that different pores may be sensitive to different numbers of nucleotides within the constriction. In this example, sequence accuracy was determined by comparing Hidden Markov Model (HMM) results using the original de Bruijn sequences as...
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