Thursday, 29 March 2012

There's more on pores than just Oxford Nanopores

Oxford Nanopores has recently presented at AGBT its new GridIon and MinIon, the first avaiable DNA sequencers based on nanopore technology, and there is a lot of hype to get hands on these new machines. Another manufacturer, Genia, has step up with a nanopore based sequencing platform promising to develop the 100$ genome.

However the nanopores have such a great potential for new applications (see this extensive review on Nature Biotechnology) that many others are entering this arena with novel methods suitable for DNA sequencing and other applications.

In one paper published on Nature Biotechnology and commented on this post, the authors combined a MspA biological nanopore with a phi29 DNA polymerase to develop a new DNA sequencing strategy. The method itself involves also the use of a DNA oligomer to initially inihibit the polymerase activity. It is quite complicated to explain in words but you can see how it works in the figure below, taken from the paper itself. The authors had successfully sequenced short oligomers, as well as short random sequences, even if they had to admit that issues remain with homopolymers (basically due to the influence of adjacent bases on the reading of the single basis moving through the pore) and the oligomer bloker. However Gundlach, ono of the authors, said the system has "the potential of being a very good reader with very high confidence and quality."

(a) Crystal structure of M2-NNN MspA. Charged vestibule residues are indicated in blue (negative) or red (positive). (b) A schematic depicting a standard experiment. Roman numerals correspond to positions in the current trace in c. (c) The measured blockage current (Ib) as a fraction of the open pore current (Io) is shown for a sample event. (i) A single MspA pore (purple) in a lipid bilayer (gray). The template strand (black) contains the sequence to be read. A primer strand (blue) is hybridized to the template's 3′ end. A blocking oligomer (red) with a 3′ end of several abasic sites is adjacent to the primer. The phi29 DNAP (green) binds to the DNA to form a complex that is driven into MspA. A positive voltage is applied to the trans side. The single stranded 5′ end of the DNA-motor complex threads through MspA and the ionic current drops. (ii) The electric force on the captured strand draws the DNA through the phi29 DNAP, unzipping the blocking oligomer. Arrows show the direction of motion of the DNA template strand. The ionic current exhibits distinct steps while nucleotides pass through the pore. (iii) The blocking oligomer is removed and DNA reverses direction (marked by blue dashed line). (iv) The phi29 DNAP incorporates nucleotides into the primer strand, pulling the template toward the cis side. The current repeats previously observed levels in reverse time-order. Two abasic sites produce a high current peak (~0.6 Io) indicated by red Xs. This marker is first seen during unzipping and then again during synthesis. When synthesis is complete, the DNA and DNAP escape to the cis volume, marked by the return to Io.

A second paper published on Nature Methods and also commented on GenomeWeb, has a more "technological" approach, based on a new CMOS sensor (see the figure belowe taken from the paper) to increase the sensibility of voltage change detection, which is at the basis of every nanopore system. Using solid-state nanopores platforms (rather than biological nanopores) they developed a current preamplifier that improves the signal recorded, allowing to take faster measurements. On of the main drawbacks of current nanopore technology is that it is necessary to slow down the DNA moving through the pore to make the voltage changes distinguisable, but "it would be nice to come closer to the natural rate at which DNA goes through the pore," said Jacob Rosenstein, first author of the paper. The speed at which weak currents through nanopores can be measured is not limited by the speed of the electronics, Rosenstein explained, but by the signal-to-noise ratio. "Whenever you try to measure something faster, you inevitably have more noise in the measurement."
Authors applied their new technology to measure the current trace of short DNA oligonucleotides of up to 50 base pairs and while they were unable to determine individual bases from the current trace, they could record the current signal with a bandwidth of 1 megahertz, about ten times faster than commercially available amplifiers.

(a) Schematic of the measurement setup. (b) Cross-section schematic of the low-capacitance thin-membrane chip. (c) Optical micrograph of the 8-channel CMOS voltage-clamp current preamplifier. (d) Magnified image of one preamplifier channel. (e) Optical image of a solid-state silicon nitride membrane chip mounted in the fluid cell. (f) Transmission electron microscope image of a 4-nm-diameter silicon nitride nanopore.

1 comment:

Jannah Delfin said...

I appreciate all of the information that you have shared. Thank you for the hard work!
φ29 DNA Polymerase responsible for the replication of the Bacillus Subtilis phage φ29. - phi29