Nanopore DNA sequencing: Precision and Control
[I don’t think this is one of my better note sets – the technology is neat, the results are fun to watch, but you can’t capture such a rich data stream in a blog… sorry.]
Two types of nanopore sequencing: Exonuclease Sequencing, Strand Sequencing.
Exonuclease cleaves bases, so you don’t move backwards, strand sequencing converts ssDNA to dsDNA so it doesn’t move backwards through the pore.
Not going to talk much about how channels work – basic idea, charge potential across membrane, resistance changes as things move through. Charge per unit area per unit time is VERY strong.
Good new: non-covalent chemistry determines currents for GATC. (non-covalent chemistry, not size, dictates current.)
History of nanopore seq.
- ( John Kasianowiz @NIST) alpha hemolysin pore to measure current. worked molecule by molecule – not single base.
- Simulation showed single bases are passing through in single file.
- Wild Type alpha hemolysin pore. Ten nucleotides contribute to pore resistance.
- convoluted by 3 “reading heads”
- Did protein engineering to modify by site directed mutagenesis till they got one that could distinguish all 4 bases. (Bayley group)
- Jens Gunlach lab: Moved to MspA. Analogy: Alpha homolysin is more like a champagne flute, where MSp has a 1nt width gap at the bottom of a shot glass like pore
- Better separation of CTA,, but AG still overlap, but far better than hemolysin.
Polymerase and nanopores:
DNA replication in a crystal (A family polymerase). [Ok, that’s cool, the crystal is still active, so you can take images over time to observe the chemistry happen!]
Sub-millisecond active control of DNA template control. Seredipitous discovery: At end of peak, there’s a voltage change IF the enzyme is departing, so it works as a good control.
Neat experiment where ssDNA is bounded by dsDNA on both sides of the pore, can watch the processes back and forth.
Tethering polymerase to pore isn’t bad – can kill pores.
Blocking Oligomers [Very graphical flow. I can’t describe this fast enough.]
Polymerase catalyzed synthesis to +12 Endpoint (Klenow fragment) and shown in a movie. Pulsing 20s intervals.
is phi29DNAP better? 2ms vs 2 seconds. Applied voltage causes phi29DNAP to tease apart dsDNA absent catalysis.
[This whole talk is images of results, by the way, nearly impossible to get much down that explains the content.]
Movie and results (Leiberman et al JACS 2010)
A ‘Branton’ test: use mix of dntp w ddATP.
Basically, all of the requirements for what’s needed to do nanopore sequencing … but lots letf to do.
- Seuqening and re-sequencing individual native DNA
- Read lengths longer than inudustry standard
- Sequencing across ethernet.