[BioKiwi]

Aerosols build in labs over time. In a post PCR area you can use ART tips, UV and gloves but this minimises contamination it won't remove it. A good PCR once 'opened' will generate aerosols with 10^5-10^9 copies of DNA that can travel through HEPA filters and build in a lab.

If I understand you correctly, you are saying that contamination by PCR is inevitable and nothing can be done about it. I have to disagree with you because PCR has been and is and probably will be corner stone of molecular biology.

If I open my PCR tube in a PCR cabinet with outside filtered air pushing through, any aerosol will be pushed away by air flow. Any possible remaining amplicon can be degraded by UV light or cleaned chemically and cabinet will be free from any amplicon. The question is what evidence you have or you can refer to show that usual lab hygiene is not sufficient to prevent PCR contamination. An amplicon that supposedly sticks to lab wall, how likely can be airborne again and land in a PCR tube. In a very slim probability if it lands how that really is going to affect results because the target amplicons will be in billions.

I strongly, would suggest external expert review of ancient DNA set up and practices if they can not control PCR contamination.

[bunce]

If I understand you correctly, you are saying that contamination by PCR is inevitable and nothing can be done about it. I have to disagree with you because PCR has been and is and probably will be corner stone of molecular biology.

If I open my PCR tube in a PCR cabinet with outside filtered air pushing through, any aerosol will be pushed away by air flow. Any possible remaining amplicon can be degraded by UV light or cleaned chemically and cabinet will be free from any amplicon. The question is what evidence you have or you can refer to show that usual lab hygiene is not sufficient to prevent PCR contamination. An amplicon that supposedly sticks to lab wall, how likely can be airborne again and land in a PCR tube. In a very slim probability if it lands how that really is going to affect results because the target amplicons will be in billions.

I strongly, would suggest external expert review of ancient DNA set up and practices if they can not control PCR contamination.

I never said PCR contamination is inevitable - there are many systems in place both molecular - (e.g using Uracil and UNG) and physical (ART tips and airflows) that minimise its potential to contaminate.

My warning was, and still is, around amplifying amplified DNA.

Using your scenario - working inside a PCR cabinet - as an example. If you open first round PCR products (to put thin into second round PCR) inside the cabinet aerosols are still an issue within the cabinet.

As some have pointed out; contamination is dependent on copy number - but in cases where 1st round PCR's have failed to amplify then any contaminating molecules can prime the 2nd round PCR. It is a risk, and people should be aware of it.

Evidence of contamination in PCR is everywhere - have you never had a contaminated PCR? - if the answer is yes then that is evidence that "control measures" you used failed. Thanks for the offer to review our lab practices - If you want to come visit our clean lab then please do;(https://jdlc.curtin.edu.au/facilities/trace.cfm) - always looking for cleaner ways to generate data.

[BioKiwi]

Thanks for clarifying your position on PCR contamination issues. I do not think that a mechanism or evidence has been provided for PCR contamination you have implied in earlier posts:

Aerosols build in labs over time. In a post PCR area you can use ART tips, UV and gloves but this minimises contamination it won't remove it. A good PCR once 'opened' will generate aerosols with 10^5-10^9 copies of DNA that can travel through HEPA filters and build in a lab.

I was looking for evidence that shows an amplicon travels through HEPA filter, is not degraded by UV exposure, builds up in the lab and somehow ends up in another PCR reaction.

[bunce]

Thanks for clarifying your position on PCR contamination issues. I do not think that a mechanism or evidence has been provided for PCR contamination you have implied in earlier posts:

I was looking for evidence that shows an amplicon travels through HEPA filter, is not degraded by UV exposure, builds up in the lab and somehow ends up in another PCR reaction.

Morning BioKiwi,

Sure, If you would like a few articles to help out with your research (it is not comprehensive - but provides a few exemplars).

I’d start with this paper;

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013042;

who comment;
"Carry-over contamination with products of previous PCR and cloning steps is one of the most serious threats for the generation of reliable results from minute quantities of DNA and also prevents the reliable evaluation of other contamination sources. The amplification and cloning of even a very small number of initial molecules produces up to 1013 molecules that are all identical and indistinguishable from those targeted. These contaminants may be carried over from previous amplification reactions due to aerosolization when the cap of a microtube is opened, and subsequent contamination of gloves, pipetting devices, laboratory surfaces, door knobs, handles of refrigerators and freezers, etc., in addition to reagents. This problem is exacerbated when semi-nested and nested PCR protocols are used. Carry-over contamination can be limited using dedicated devices, physical separation of the different experimental steps and stringent experimental procedures [51]. Used alone, these methods cannot guarantee complete protection [44], even when used in contained laboratories. Indeed, DNA is mostly spread by the experimenters who can be repeatedly contaminated by previous PCR and cloning products. These products can remain on many surfaces for long periods if they are not systematically identified and decontaminated after each potential contact with PCR products.”

and

"There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration”

UV clearly works to modify DNA but - even in hoods generates a low of shadows - it is not a ‘complete’ solution; see above and;

Comparison of the effects of sterilisation techniques on subsequent DNA profiling. http://www.ncbi.nlm.nih.gov/pubmed/17318649


HEPA - the physics of these filters are;

“DNA segments less than 300bp measure under 0.1 microns and pass through HEPA filters (0.3 micron pore size) in biological safety cabinets and are not eliminated by UV light or many disinfectant solutions.” - PCR for Clinical Microbiology: An Australian and International Perspective.


maybe finish with this paper on building clean labs and organising workflows.;

http://www.sciencedirect.com/science/article/pii/S0940960211000732

PCR may be, to borrow your terminology “the corner stone of molecular biology” but within NGS workflows it is a necessary evil; bias, contamination, chimeras, error. You can go about designing workflows to minimise its impact - but " control" is an altogether different claim especially when amplifying amplified DNA.

If you want some more feedback on PCR contamination within NGS workflows maybe start a new thread on that topic? - I have aired my thoughts on the risks - the reality is that a lot of data on contamination does not end up in publications.