My lab was having a discussion on which type of PCR to do to best amplify our 16S genes from our genomic DNA extractions. After my mentor explained what nested PCR was I recommended it based on this thought: If we initially amplify using primers for the full 16S gene followed by amplification with primers for the specific hypervariable region of interest, it would nearly eliminate the contamination/background noise from incomplete 16S genes (such as those ubiquitous in the environment or extraction kit reagents), which would mean any remaining sequences would have to have originated from intact bacterial cells either from our samples or from bacterial contamination from the person or hood.
I can try to clarify better if any of this did not make sense. I am asking here because it made sense to us logically but my PI asked me to look into the literature, and I haven't been able to find anything that would validate this; it seems like it would be difficult to demonstrate experimentally.
EDIT: The question is essentially is it safe to assume that all remaining sequences after the nested PCR were derived from viable bacteria, not just exogenous DNA fragments?
Nested PCR is generally employed to improve the sensitivity. What I understood from your statement is you are initially amplifying using primers for the full 16S gene followed by amplification with primers for the specific hypervariable region of interest. As your PI explained, the 16S rRNA gene is a section of prokaryotic DNA found in all bacteria and archaea. To eliminate the contamination/background noise from incomplete 16S genes, in the second step you are using more specific primers targeting hypervariable region.
You can read this article for further details. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562909/)
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