When I did a PCR reaction using the template DNA of mosquito using the outer primers F3 and B3 I found my target region of interest amplifying at an annealing temperature of 55 degrees. But when I do a LAMP at 55 degrees and even at 65 degrees I am getting similar bands in NTC and my sample. I am not able to confirm whether my product got amplified or not. So I would like to know how do we determine an optimum temperature to carry out LAMP?
Amplification in negative would be due to contamination and not due to problem in temperature. LAMP with bst polymerase works best at temperature between 60-65 degree Celsius.
Varsha Venkatesh I have a doubt. Lets say primer dimers are being formed in NTC, will the pattern formed by primer dimers be the same as the ladder like bands obtained in the sample.
No madam it would not be the same, primer dimers are always below the last band of the ladder, if you obtain bands in negative control it tells that there is contamination, primer dimers never form a band like appearance
You can still discriminate your true amplicon from a false-positive amplicon. For that, you just need to design a "fishing" probe that is complementary to one of the loops produced during LAMP and implement magnetic particles pre-functionalized with such a probe to "extract" your amplicon in a sequence-specific way. You can also find more information about our approach as well as summary of what other people did to varify their amplicon sequence in our recent paper: "Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2".
In fact, the optimal temperature for LAMP is related to the enzyme and not to the primers. The enzyme works better at 60-65°C so that's why LAMP assays are conducted in that temperature range. And about your NTC that show bands, it's related to contamination and not to sub-optimal temperature. The optimal temperature will be the one that gives the best results of amplification, in other words, the most beautiful bands on your gel :)
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