I'm familiar with the principles of primer design and have previously designed primers for use in modern DNA. However, in my current project I am working with ancient DNA (nuclear gene) and need to design primers to work in this context - I am wondering if there are established additional considerations that need to be taken into account.

What I have discovered so far is mostly literature dealing with the damage patterns in ancient DNA (i.e., amplicons tend to have damage at the 5' and 3' ends of each fragment, so sequence data from the centre parts of fragments are more reliable). I have seen the suggestions that multiple, overlapping primer pairs might be used to detect low-copy number fragments, but this suggestion, while useful, doesn't tell me much about the design of the individual primers.

My own thoughts are that since ancient DNA is typically fragmented into ~150 bp (depending on age - my samples are around 250 years old), multiple primer pairs might help. My target is around 500 bp.

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    $\begingroup$ Why not contact some of the labs that do a lot of old DNA work (not sure 250 years counts as ancient :) ) and ask them for advice or pointers to discussions of primer design. Since it seems samples vary widely in their degree of preservation, perhaps they will have suggestions on how to evaluate the quality of your specific samples. $\endgroup$
    – Armand
    Aug 9, 2021 at 18:36
  • $\begingroup$ That's a great suggestion, thank you! I agree that calling 250-year old samples 'ancient' sounds and feels odd, but as of yet there's no distinction between "kind of old DNA" and "really old DNA". $\endgroup$
    – natb
    Aug 9, 2021 at 23:53
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    $\begingroup$ @Armand 250 years is a lot of time for DNA, which is why it is commonly referred to as ancient. I agree that the term is misleading, but it is frequently used. $\endgroup$
    – Roger V.
    Aug 14, 2021 at 5:31
  • $\begingroup$ @natb could you link the literature you reference? I assume you've read this paper on ancient DNA damage and this paper on amplification of ancient DNA? $\endgroup$
    – acvill
    Nov 7, 2021 at 18:22
  • $\begingroup$ @acvill Yup, I've read both those papers. Unfortunately that's the closest I've gotten to finding the information I'm looking for. I can link other sources I've read but they are all less relevant. $\endgroup$
    – natb
    Jan 28, 2022 at 18:47


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