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I have been given a project involving a plasmid that contains long stretches of Adenine (60 or 120 bases each). These PolyA stretches are interrupted by the occasional G or C.

I understand that performing PCR, cloning, DNA synthesis, mutagenesis, and sequencing on repetitive sequences is difficult. What techniques and practices should I follow to improve the accuracy when dealing with these PolyA sequences?

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  • $\begingroup$ What exactly are you trying to do with this plasmid (more spef. this PolyA tail). Do you need to amplify it, digest it etc... depending, different strategies can be implemented to minimize error with the polyA tail $\endgroup$ – Adam Radek Martinez Feb 13 '18 at 2:49
  • $\begingroup$ @AdamRadekMartinez Let's say I wanted to replace those interrupting G's with an A. How does site directed mutagenesis work when you have more than 1 potential site? How do you sequence the DNA to verify that the mutation worked? $\endgroup$ – user137 Feb 13 '18 at 11:04
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You can use much longer primers (up to 70bp or so), such that they span the repetitive region you want to change and some more specific sequence to anchor the primer (assuming your vector allows). If the multiple sites you want to change are within range of one another, you can order primers with 2 or more mutagenised sites - though expect your efficiency to drop. Your other option would be to do multiple rounds of mutagenesis, altering one position at a time.

I would then simply try these primers on a gradient PCR using a good enzyme like NEB's Q5. Trying to find a temperature for the correct size band will likely be key.

The real problem you're going to have though probably isn't the cloning, it'll be proving it. Homopolymer stretches are a real problem for sequencing, and the longer they get the worse it is. Automated sequencers find it difficult to tell between, for instance, 29 Adenines, and 30 adenines in a row. When your sequencing results are returned, if your sequencing is off by a base or two, it will be pretty much impossible to tell if your sequencing was wrong, or your construct was.

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Okay, one of the main problems with polA tails is with PCR, which is a method commonly used in molecular cloning, specified mutagenesis etc...

One problem with PolyA tails and PCR is with the primers. Since A's only have 2 hydrogen bonds, instead of 3, this can make it hard to design good primers in this area. If you want to amplify this region using PCR, you may have troubles designing primers since it is recommended that you have a lot of G and C's (espically near the ends for stability). Thus, creating blunt ends, mutagenesis etc will be more difficult.

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