Poor Primer Selection

What would cause a sequencing primer to anneal in the correct site, but extend in the wrong direction? The end result was compliment to the previous sequence from which the primer was chosen.

Summary

The researcher was baffled with the result. It was a primer-walking project to complete the sequence for a 2kb insert. The first sequence was performed using universal M13(-21) and produced a clean 600 plus base read length. The primer was selected 450 bases downstream and was expected to continue in the same direction. Instead the result was a complimentary sequence covering the same area as previously sequenced (see figure).

Resolution

The primer selected consisted of 20 bases. Seventeen of the bases were directly complimentary to each other forming a primer that was essentially reversible. The primer simply annealed to the same region of the reverse strand of DNA predominantly matching the 17-base compliment. The resulting sequence was very clean showing no hint of a problem. The cause was simply poor primer selection. Nowadays primer orders are generally submitted on-line and include automated evaluation of secondary structures in the primer so this problem shouldn’t happen anymore. This was an interesting phenomenon to see.

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