PCR bias by backfolding amplicons

Sebastian123 · 02-04-2011, 07:41 AM

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.

ECO · 02-04-2011, 08:30 AM

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.

parthiban · 02-09-2011, 03:42 AM

Hi Sebastian / Eco, do you know a tool/software that calculates a score for amplification efficiency....taking all (or critical) issues into account?

Sebastian123 · 02-09-2011, 04:10 AM

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.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
The NEXTflex™ PCR-Free DNA Sequencing Kit Reduces Bias in NGS Library Prep	Bioo Scientific	Vendor Forum	10	12-20-2012 08:10 AM
PCR primer -- a PCR simulation on your computer	gringer	General	0	01-26-2012 03:03 AM
RNASeq removing PCR bias	schaffer	Bioinformatics	3	10-20-2011 07:00 PM
Number of PCR How many PCR cycles to enrich adapter-modified DNA fragments	MGH Man	Sample Prep / Library Generation	5	07-26-2010 06:15 AM
Nested PCR amplicons	AEscobar	454 Pyrosequencing	4	07-01-2010 07:55 AM

02-04-2011, 07:41 AM	#1
Sebastian123 Junior Member Location: Germany Join Date: Feb 2011 Posts: 2	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.

02-09-2011, 04:10 AM	#4
Sebastian123 Junior Member Location: Germany Join Date: Feb 2011 Posts: 2	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 04:23 AM.

02-04-2011, 08:30 AM	#2
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	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.

02-09-2011, 03:42 AM	#3
parthiban Junior Member Location: Cambridge Join Date: Jan 2010 Posts: 1	Hi Sebastian / Eco, do you know a tool/software that calculates a score for amplification efficiency....taking all (or critical) issues into account?