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Thread: Using speed of polymeraze to sequence DNA?

  1. #1 Using speed of polymeraze to sequence DNA? 
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    There are considered some approaches to sequence DNA base by base - for example by making it go through nanoscale hole and measure its electric properties using some nanoelectrodes.
    Unfortunately even theoretical simulations says that identifying bases this way is already extremely difficult ...
    http://pubs.acs.org/doi/abs/10.1021/nl0601076

    Maybe we could use nature's ways to read/work with DNA?
    For example somehow mount polymerase or ribosome and somehow monitor its state...

    I thought about using speed of process to get information about currently processed base.
    For example DNA polymerase to process succeeding base has to get from environment corresponding nucleoside triphosphat - there are only four of them - we can manipulate their concentrations.
    If we would choose different concentrations for them, there would be correlations between type of the base and time of its processing - by watching such many processes we could determine the sequence.

    Is it doable?
    What do you think about such 'base by base' sequencing methods?
    How to use proteins developed by nature for this process?


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  3. #2  
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    Secondary structure of the unfolded DNA, base stacking, and other non-active-site sequence factors will affect rate.


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  4. #3  
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    Yes, there are a few random factors which would have some small influence on this speed and still it's stochastic process - sometimes it will quicker catch 'nucleotide carrier' of smaller concentration ... its why it would be required to process given ssDNA a few times and use some Bayesian analysis to determine the sequence.
    Still it should be many degrees of magnitude faster than e.g. pyrosequecing in which each nucleotide requires macroscopic time cycle.

    To reduce such effect we could somehow mount straightened DNA and choose really large differences in concentrations (even like 1:10:100:1000)
    The movement of polymerase could be watched optically (for example by attaching to it something producing light, like luciferase and surround the space with CCD).
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  5. #4  
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    Why not just use the dideoxy method

    Tridimity
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  6. #5  
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    Standard method like Sanger You mentioned or pyrosequencing are expensive and slow.
    For many purposes we would like to have a method which can sequence the whole human DNA in days at cost like a few thousand dollars ... for that we need a completely different approaches - like trying to determine base sequence while ssDNA passes through some nanopore/protein/ribosome ... in such approach to process a base there is needed not macroscopic time like in pyrosequencing, but microscopic one - it would be many orders of magnitude faster.
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  7. #6  
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    Quote Originally Posted by Jarek Duda
    Yes, there are a few random factors which would have some small influence on this speed and still it's stochastic process - sometimes it will quicker catch 'nucleotide carrier' of smaller concentration ... its why it would be required to process given ssDNA a few times and use some Bayesian analysis to determine the sequence.
    Still it should be many degrees of magnitude faster than e.g. pyrosequecing in which each nucleotide requires macroscopic time cycle.

    To reduce such effect we could somehow mount straightened DNA and choose really large differences in concentrations (even like 1:10:100:1000)
    The movement of polymerase could be watched optically (for example by attaching to it something producing light, like luciferase and surround the space with CCD).
    A bayesian analysis will not allow you to distinguish a reproducibly slower rate due to active-site-dependant-slowing from non-active-site-dependant slowing, you need knowledge of the sequential context.

    I am not clear how you will 'straighten' DNA in a mounted fashion.
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  8. #7  
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    And so to make the process more predictable, there would be probably this 'straightening'/unfolding needed...
    In 15min I thought about three ways ... so probably there are also some better ...

    The best looking would be using some nanopore or a membrane protein through which there came 'locally straightened' ssDNA and such that we can control speed of this process, for example by changing electric field. Such nanopores already works.
    We would use it to make that the polymerase works near this pore (towards it) and so on unfolded part.

    Another ways to straighten/unfold DNA could be just mechanical - for example attach it on one end and pull it for example into a capillary ...
    Or attach oppositely charged ions on both sides and use external electric field ...

    ----------
    Unfortunately measuring position of polymerase is in fact really difficult ... optical methods are rather not precise enough ... making many snapshots using electron microscope could damage it ...
    Alternative approach is attaching polymerase to cantilever of atomic force microscope - it should 'feel' single steps it make ... so when we would use large differences in concentrations, times between steps would very probably determine base. Of course we would have to process given strand a few time to get required accuracy.
    There is another discussion if someone is interested: http://www.scienceforums.net/forum/s...d.php?p=534170
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  9. #8  
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    An exciting development, Flusberg et al have developed a method for sequencing DNA and mapping epigenomic methylation patterns, based on the kinetics of DNA polymerase. The technique relies on the incorporation of fluorescently-tagged nucleotides, which upon incorporation into the growing DNA strand produce a flash of light. The colour of the light corresponds to the identity of the base. Analysing the pulses of light and the time intervals between them, can also allow you to discern whether methylation is affecting DNA polymerase activity.

    More can be found in this week's Nature Methods

    http://www.nature.com/nmeth/journal/...meth.1459.html

    Best,

    Tridimity
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    Would such a system be faster than technologies like the 454 method? 454 can do about a billion bases per day, for example. A method like you suggest (even if the considerable hurdles were able to be overcome) would seem to an inherently slow process.
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  11. #10  
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    Dear Zwirko,

    I'm not sure how fast it is, one drawback is that at present, the technology only has a methylation readout of ~1,000 bases.. although it has been stated that the cost of a full-length genome methylation map would drop to $100-1,000

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  12. #11  
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    Sorry tridimity, I should've quoted the person I was responding to, rather than just leaving my disembodied comment floating around like that. It was Jarek's idea that I figured as being too slow. Sorry about that.
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  13. #12  
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    A new development in the area of developing relatively affordable, high-throughput sequencing technologies... Rothberg et al report this week in Nature a novel technique that relies on the ability of chips to sense protons released as nucleotides are incorporated into cDNA. The $49,500 device can sequence a bacterial genome in a couple of hours... still a way to go in terms of reaching the $1,000 goal and increasing speed but surely a feat in sequencing technologies.

    sequencing.jpg

    Above: Sensor, well and chip architecture taken from Rothberg et al (2011).

    Actually becoming quite excited about this... affordable, high-throughput whole genome sequencing seems possible in our generation!

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