I'm now operating an experiment of MT genes amplification and sequencing. When amplifying the mt large subunit ribosonal RNA gene (16S), the agarose gel electrophoresis result showed me that there existed a smear bigger than the aimed band. Everytime when I was sequencing this gene I may encounter the same problem. So who can tell me what's the smear's component, and why it was mistakenly amplified?
(I use 16SAR/16SBR as the PCR Primers)
I think that the weaker, upper band is not a mistake but indicates that your amplified sequence has at least 2 sequences (snps or small indels).
Your amplification is very strong and if we have 2 very similar sequences then at the last cycle of pcr a lot of amplified product will anneal to other product molecules. If we call these amplimers N and M where sequence M differs from sequence N by one base then when the 2 sequences combine we get NN and MM and some MN. In an agarose gel NN and MM are the same size so run as one band but NM has a mismatched base forming a bubble in the dna and this changes the shape of the dna so it runs as larger than N N and MM but the heteroduplex band just runs at a slower rate through the gel so is interpreted as a larger size band. To test this idea you can cut out the smaller stronger band and denature it,reload it and you should once again generate 2 bands just like the picture you posted. Sequencing either band should also show that you have 2 sequences correctly showing in both bands
I see this often with universal 16S mtDNA primers. I have sometimes rerun the PCR with slightly higher annealing temperature, increased by a degree or 2 degrees. on the other hand if you want to try sequencing the PCR products this can work as well, and in my experience the non-target band did not cause any detectable issues
@Brenden Holland Yes, my primers (16SAR/16SBR) are universal primers. I noticed that too. A higher temperature may let the non_targeting band paler. However, this strategy may also lower the efficiency of amplification, i.e. causing amplification failure of other samples. Till now, my method is to send the original product to sequencing companies and to ask them to purify it. However, once a sequence was longer (100bp) than other sequence results, which I then realized may be the non-targeted. BLAST show that it is most similiar to 16S, but it can't be properly aligned with 16S sequences because of lots of insertions. I guess maybe there's a fake gene that have some parts same as mt 16S, causes the non-targeted amplification?
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