Good day everyone. I'm working with eDNA Metabarcoding for freshwater fishes in Brazil, I ran tests amplifying positive controls and samples with the primer MiFish-U (Miya et al 2015) alone (no overhang sequence, no tags, no adapters) and got good results, but when I started doing the same protocol, and some more with modifications in MgCl2 concentration and T° cycles, using the same primer with the Illumina adapter and a tag (overhang sequence), I got less than 10% successful amplifications.
According to the literature, overhang sequences shouldn't have a significant effect on the PCR protocol, does anyone know why is this happening?
Mg ions will change the annealing temperature of primers (downwards if you are increasing the Mg amount) but adding a tag which does not anneal to the target dna will not make a difference so long as you do not include the tag sequence when calculating the annealing temperature which must remain (or be lower) than your original Ta because only that part of the primer can anneal to the target dna
Hi Ricardo,
Not sure why it is not working for you but I have previously used the MiFish primers with some quite large overhanging sequences and did get good amplification. Generally, adding overhanging sequences will reduce the amplification efficiency slightly so maybe increasing the number of PCR cycles might work. Alternative, overhanging sequences could in some cases influence primer interactions (i.e. formation of secondary structures) would can further reduce PCR efficiency or in extreme cases inhibit amplification completely.
I would recommend you to perform some tests on a few known positive controls (DNA extracted from fish tissues) to check the impact of various conditions on your amplification success when using the Mifish primers solely or in combination with overhanging sequences.
Best of luck
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