Usually for sensitivity, researchers will be using cloned DNA. So that we can test the sensitivity based on DNA copies. However, is there any difference if I'm using purified DNA from PCR product?
Dear Shazleen, it depends upon the precise nature of your PCR product - is the same as the region for the LAMP reaction. How do you intend to generate your PCR product for testing the LAMP reaction on? If the product is only just larger than the LAMP region then generally LAMP will work better on Plasmid rather than PCR product. This is because the upstream and downstream sequences in the plasmid or genomic DNA appear to facilitate the DNA entering into dynamic equilibrium which is essential for the displacement primers to get in and initiate the displacement of FIP and BIP derived products. Are you intending to amplify RNA or DNA in your assay?
Dear Dr Polley,
Yes, my target gene for PCR and LAMP is the same.
Actually I want to conduct limit of detection, compare between PCR and LAMP. Precisely, what I am doing now is purified the gel of my PCR product and read the DNA concentration. Based on the concentration, I will conduct a serial dilution and follow by PCR LAMP screening, to find out the detection limit. I intend to amplify DNA sequences.
As you know, there are a lot of studies conducted their limit of detection based on the copies of DNA that have been cloned in plasmid. Do you think my detection limit test is valid if I am doing this method?
Reply from you is really appreciated.
In my experience which is mostly RT-LAMP the qRT-PCR often exceeds the RT-LAMP limit of detection by up to 1 log. For regular LAMP (e.g. with bacterial targets) we often see the LAMP getting down to
Dear Shazleen
which primers are you using to generate your DNA, B3 and F3 or are you generating a larger fragment. As I said LAMP sometimes works better if it has a larger upstream region. If you are panning a diagnostic test the only true way to calculate LOD is with a real diagnostic sample as far as LAMP goes and PCR fir that matter because clean up is a key and integral part of the test. You'll just have to see what happens with the PCR product and bear in mind what I've said. Cloning PCR products nowadays is a very simple matter thanks to vectors such as Pgem T easy from Promega. You can achieve almost a 100% success rate. Robert had given you very sound information aswell.
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