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.
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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