check LAMP! pcr is soooooo 90ies..

[Marc Dusseiller]

hei garage biologists,

just saw some interesting low-cost stuff at a conference in kenya. you know about LAMP? seems like a great and cheap alternative to pcr, especially on the instrumentation side.

http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0000147

more stuff from low-cost diagnostics soon,

marc

[Cathal Garvey]

LAMP is an awesome method for on-site PCR, but (speaking as someone who
*hasn't* tried it) seems to be harder to optimise and design for
general-purpose DNA amplification.

That is, standard PCR is better for prototyping a reaction, but for
creating a protocol for general or regular use, LAMP is probably a good
next step; as you point out, it requires little in the way of
instrumentation, and because there's no cycling of temperature, that's a
huge source of experimental error removed! Awesome for walk-by workshops
for DNA testing etc..

--
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twitter.com/onetruecathal
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PGP Public Key: http://bit.ly/CathalGKey

[Pieter]

The papers I've seen on LAMP look very promissing. Especially this one on malaria: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013733

I'd like to read some more about the inhibitory effects that have been described when samples are not completely purified. I can imagine that 6 primers make the reaction more sensitive to polutants. Also the economic performance of the method is unclear to me (e.g. how much does it cost?).

Does anyone have a good reference to an article where these parameters are studied? I'd love to learn more about it!

[Nathan McCorkle]

Cost is comparable to PCR, 3x amount of primers, Bst polymerase from NEB, isothermal temp controlled device

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[Cathal Garvey]

> isothermal temp controlled device

= Cup of Coffee? :)

>>>> http://www.plosntds.org/**article/info%3Adoi%2F10.1371%**
>>> 2Fjournal.pntd.0000147<http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0000147>

>>>>
>>>> more stuff from low-cost diagnostics soon,
>>>> marc
>>>>
>>>
>>>
>>> --
>>> www.indiebiotech.com
>>> twitter.com/onetruecathal

>>> joindiaspora.com/u/**cathalgarvey<http://joindiaspora.com/u/cathalgarvey>

[Ravi]

Very cost effective/sensitive. You can use 6x primers, and reaction setup is same as regular PCR except you use Bst polymerase. You can also check for product in the tube by adding a dsDNA dye such as SYBR green or even SYBR safe - visually (!). Most importantly, you only need a isothermal incubation - so no cycling. 

2 problems:

1) everything remotely connected to LAMP is heavily patented. Specific assays, reporting mechanisms, devices etc. 

2) cannot replace PCR as you are amplifying a "chain" DNA structure, not your target. So its great for diagnostics but no application to building plasmids etc.

Ravi

[Nathan McCorkle]

On Wed, Jul 4, 2012 at 1:50 AM, Ravi <sheth....@gmail.com> wrote:
> 2 problems:
> 1) everything remotely connected to LAMP is heavily patented. Specific
> assays, reporting mechanisms, devices etc.
> 2) cannot replace PCR as you are amplifying a "chain" DNA structure, not

a chain structure???

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Pieter]

The result is a continuous loop structure: http://www.youtube.com/watch?v=5Wi-kkSFy48

The patent situation is also what I see as the main limitation for it to be applied as a true POC diagnostics method. I also think the need for purification of the DNA, either by qiagen kits or boiling and centrifugation is a major drawback.

There have been studies before on how to modify the polymerase in such a way that it still works regardless of common PCR inhibitors: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655666/. I assume the Bst Polymerase has not been designed in the same way. Also NE Biolabs does not make any statement on inhibitors on the FAQ site: http://www.neb.com/nebecomm/products/faqproductM0537.asp

Reducing the amount of steps of the procedure by boiling the sample including the reagents is also not possible, since the Bst Polymerase becomes heat-inactivated at temperatures greater than 72 C...

[Ravi]

By chain I mean the accordion folded strands of DNA, the "continuous loop structure"

http://loopamp.eiken.co.jp/e/lamp/principle.html

You can get around purification concerns through additives like Chelex that remove inhibitors or even microwaving (http://genome.cshlp.org/content/3/6/365.full.pdf+html). This paper is from '94 so I am sure there are many other methods (e.g. magnetic bead purification) etc.

Once you can get past this though, LAMP looks very awesome, check out the results that can be determined visually. I could imagine a simple photodiode being more than sufficient for detection.

http://www.hindawi.com/journals/jtm/2012/340538/fig3/

Purification of DNA is a concern but I think you can get around this. For example 

On Wednesday, July 4, 2012 2:39:22 AM UTC-4, Nathan McCorkle wrote: