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Friday, February 26, 2010

AGBT 2010 - Jonas Korlach - Pacific Biosciences

Direct Single Molecule, Real Time RNA Sequencing.

Opportunity to further work with this platform to replace enzyme in ZMW with other enzymes of interest - can observe new functionality.

"Single Molecule Realtime Biology" [SRMB? How do you say that acronym?]

Of interest: Reverse Transcription
* replace polymerase with rna polymerase (reverse transcriptase)
* have done this - simple extension tests.
* done kinetic analysis, and the phospho dntps are incorporated well, but MUCH slower (1 order slower) than non-marked nucleotides

Tested the system out anyhow.
* Seems to work in principle - albeit it's slow. One dNTP in enzyme is not yet one nucleotide inserverion.

Ribosomal RNA Sequencing.
* Can withold catalytic metal, which allows binding, but not ligation. Thus, you can just watch the flourescnece - and in this case, binding only happens with correct nucleotide.
* can also detect modified RNA bases - eg, Pseudouridine. Can measure binding time - takes longer.

Detection of Modified RNA bases
* pauses indicate kinetic changes

For viruses, you can get a single enzyme to process the entire genome of a virus - very long read lengths at the tail end of the distribution.

HIV reverse transcriptatse translocation dynamics.
* use terminating bases and AIDS drugs - and monitor incorporation and pulses.
* Show graphs of kinetic analysis of P-Sites and N-site
* Can then study binding in the presense of the terminators/drugs.
* Can calculate binding energy from puslses.

* Demonstrated SMRT RNA sequencing - still room to grow.
* Deomnstrated SMRT Biology - Translation (shown tomorrow) and reverse transcriptase.



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