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Sanger's PCR chain-termination sequencing, by its theory, is a simple method to sequence DNA from organisms. But what will happen if there are 2 allelic DNA with same primer binding site, but variations in some-other part?

My question is; 1. Is such overlap practically happens? 2. If it happens, then what problem it creates? (or does it create any problem at all); and 3. if it is problematic, then what techniques used to avoid or reduce such noise?

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  1. Yes, this can happen if you are not checking your primers against the reference sequences.

  2. Your PCR is done from two different pieces of DNA leading to two sets of chain-terminated fluorescence marked DNA. This leads to superimposed sequences, which looks like this and where you cannot differentiate, which sequence belongs to which strain (image taken from here):

enter image description here

  1. It is problematic, the solution is to design new primers (which are located slightly outside the old target sequence) which only bind to one specific sequence. You cannot purify only one product, as you cannot discriminate between the two.
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