I am trying to optimize loop mediated isothermal amplification (LAMP) conditions that favor a specific bacterial species. Each reaction contains FIP/BIP, F3/B3, and LF/LB primers, dNTP, amplification buffer, as well as Bst. 2.0; concentrations based on related publication. My positive control is set at 1000 CFU/reaction, has inconsistently appeared on my 1.5x gel. When I prepare a LAMP mixture for 1-10 reactions the results are strong, however, when I run 15-96 sample reactions simultaneously, the positive control is inconsistent. I tried thoroughly mixing the LAMP mixture without any positive gain. At first I thought I was exhibiting some type of human error or overall mistake but at this point I made sure to rule that out. I have even tried to denature the template DNA prior to reaction to ensure amplification, however, my results were stronger without it.
Betaine is an enhancing agent for LAMP reaction. It reduce the secondary structure formation in G-C rich template and also help Bst polymerase in strand separation.
I suggest you add 1M of betaine in your reaction without LF/LB primers. Also remember to use separate working bench and pipettes as LAMP very easy to get contaminated. Good Luck!
Hi all,
I am currently conducting a research on the primer design as many LAMP users find it difficult to get their way around getting a validated and optimised functional primer.
I was wondering if their are any assay that are any assay that are optimised and validated ready to use assays/primers for specific target. would it not safe time and add to the convenience besides getting a functional primer ?....
I have found betaine to be completely unnecessary in some cases. Probably more important if you have a highly structured target. Caveat, I am usually doing RT-LAMP for ssRNA targets.
Going back to very old LAMP literature, c. 2001, they showed denaturing prior to amplification was not necessary. I think at the temperature of the reaction the templates "breathe" a little bit, forming transient bubbles that allow primers a chance to anneal. And the strand displacing polymerase takes care of the rest.
That said, some primer sets are better than others - you can see 2 orders of magnitude difference in sensitivity, and a large difference in speed, depending on the primer design. It is hard to discern from software what is good and what isn't good, so it's more a matter of getting a few good candidate sets and trying them all.
Hi Robert, Which paper you are referring to? Did you try to catch the advantage of 'breathing effect' of DNA at 60deg.? Am curious, please update!
This is probably the one that Robert was referring to:
http://clinchem.aaccjnls.org/content/clinchem/47/9/1742.full.pdf
Clin Chem. 2001 Sep;47(9):1742-3.
Loop-mediated isothermal amplification reaction using a nondenatured template.
Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T.
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