Confusion about PCR

I seem to be getting two different answers from my book and my teacher about PCR.


1. Does PCR require a double stranded piece of DNA that you are tryig to replicate, or does it require a circular piece of DNA (i.e. a plasmid)?


I believe that when we were doing the experiments, we used a plasmid.
2. Also, I can't seem to find a source which explains how PCR works when you are also introducing a mutation at the same time.


We used different primers to introduce a mutation during the PCR, but I am still very confused as to how the primer could bind when the nucleotide sequence is not exactly matching.





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> For PCR, does PCR require a double stranded piece of DNA that you are trying to replicate, or does it require a circular piece of DNA (i.e. a plasmid).
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Circular DNA is not required. Double-stranded DNA is, but keep in mind that if you start with a single-stranded DNA that (i) can be primed by an appropriate 'foward' primer and (ii) contains the reverse complement of a 'reverse' primer binding site (**i.e. contains the sequence of a reverse primer, see comments**), then during the first cycle of PCR, a double-stranded DNA will be created from the single-stranded DNA.


In fact, circular DNA, while generally a viable substrate for PCR, is less good than linear dsDNA under some extreme conditions, especially if it highly supercoiled, because strand displacement by DNA polymerase becomes more difficult.



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> Also, I can't seem to find a source wheich explains how PCR works when you are also introducing a mutation at the same time. We used different primers to introduce a mutation during the PCR, but I am stilll very confused as to how the primer could bind when the nucleotide sequence is not exactly matching?
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> 
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The thermodynamics of primer binding is a complex but fairly well-understood subject. Each nucleotide in the primer that matches the target DNA sequence will provide favorable enthalpic (and entropic) interactions with the target, favoring binding. These favorable interactions are even more favored if the nucleotide's neighbors in the DNA chain also match. There is a general entropic penalty for binding because the degrees of freedom in a single dsDNA molecule are way lower than in two ssDNA molecules. Essentially, once there are enough matching sites between a primer and a target DNA, binding will be favorable (and this transition point will depend on temperature, with higher temperature always dis-favoring binding). The presence of a few mis-matches doesn't mean a primer can never bind, it means that the temperature at which the hybridized primer/dsDNA pair dissociates (the "melting temperature") is lower than for a fully-matching primer.


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In terms of a specific answer to the questions you have asked, Curt F. has provided an excellent answer. I just want to provide you with a couple of extra resources to start you off reading about PCR mutagenesis. 


Pages 3 and 4 of this manual give an intro to QuikChange plasmid mutagenesis using PCR and primers with small mismatches: <http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf>


This page gives an intro on introducing restriction sites to the end of a DNA sequence being amplified by PCR: <https://www.addgene.org/plasmid-protocols/PCR-cloning/>


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