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Old 02-04-2011, 06:41 AM   #1
Sebastian123
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Default PCR bias by backfolding amplicons

Have anyone of you experience to what extend backfolding of amplicon templates generated by a PCR induces a bias during the PCR procedure?

We are currently developing an experiment to identify and analyze PCR and sequencing errors by pyrotag-sequencing. I'm a computer scientist and therefore not that familiar with PCR chemistry and kinetics. I'm wondering now that if a PCR product forms a secondary or tertiary structure (by backfolding) wouldn't that cause a abortion of the amplification of this product (and by that producing only a partially amplified product). If that is the case, at which rate does this happen (e.g. if a sequence has a free energy of the thermodynamic ensemble of above a given threshold, then a amplification stop is most likely). I've already searched the literature but didn't found anything related. Maybe this is an under-investigated side effect?
Is backfolding a problem in PCR at all?

Any note or references on this topic is welcome.
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Old 02-04-2011, 07:30 AM   #2
ECO
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I think the term you are looking for is PCR "suppression"; that will probably help your literature searching.

It's typically caused if you put the same adapter on both ends of a fragment, the resultant panhandle is more stable than the properly primed event and thus those fragments don't amplify well or at all. People use this to suppress or select for certain events...so it can be embraced in some situations.
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Old 02-09-2011, 02:42 AM   #3
parthiban
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Hi Sebastian / Eco, do you know a tool/software that calculates a score for amplification efficiency....taking all (or critical) issues into account?
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Old 02-09-2011, 03:10 AM   #4
Sebastian123
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No - i dont know of any such tool and i doubt there will be-due to several reasons. This is a very difficult task to aim for and also i got the feeling all these negative side effects are constantly ignored because of their hard to address nature.

Edit: maybe i was a bit to fast. There are some tools which are trying to simulate a PCR in silico and others for Primer design and PCR setup (like melting temperature). But i dont know of any tool for calculating PCR efficiency (this is besides very subjective). But all those tools are limited in one way or another, or not free for use. I dont have a list of those tools on hand but can give you some links on request.

ECO - the term you gave me ("suppression") helped in literature searching but did not really covered my topic. I'm not interested in special PCR (like the suppression PCR) and also the setup is a complete different one. Those suppression effects are mostly related to those Headloops conformations in the present of the so called infernal terminal gaps (which are strictly bound to one-sided PCR primers).

My question is more theoretical. If you amplify DNA as part of the 16s rDNA then those fragments will have strong tendencies to form conformations and by that will be suppressed - despite the primer setup or anything else. This may induce bias as also favor other negative effects like chimeric formations and so on. My question now was if any one of you has experience with this kind of suppressed amplification and or know corresponding literature.

Last edited by Sebastian123; 02-09-2011 at 03:23 AM.
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