Hello,
I am working on a diagnostic LAMP assay and to optimize it, I have been using:
1. Synthetic wild-type DNA for my gene of interest
2. Synthetic mutant DNA for my gene of interest
I know the limit of detection of the assay with synthetic DNA, which is a bit under 5.0x10^4 copies per 25 uL reaction.
I have found that the limit of detection for purified human genomic DNA (not synthetic) is much lower, even when controlled to have the same copy number of my target gene. I have used BLAST and there are no other places on the genome for my LAMP primers to anneal.
Does anyone have any ideas as to what the difference(s) could be between synthetic DNA and purified human genomic DNA which could make it so much more available to amplify in a LAMP reaction? If anything, I feel as though it should be the opposite because with synthetic DNA there is no other DNA around to get in the way of primers that are trying to anneal...
Thanks for your ideas!
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