I'm designing a new reporter cassette to build a cell line, and I want to get this reporter by gene synthesis service.
However, I got reply from the service company that since my sequence have repetitive sequence, they cannot synthesize it in the linear DNA fragment.
I heard repetitive sequence might be critical especially for the PCR, so I want to find another design. When I checked my sequence with the repitetive sequence finding tool, I found two from the SV40 promoter region in my cassette and I can handle this part.
The problem is I found another repeats inside the antibiotic resistance gene part. Although it's just a short repeat (about 10bp), I'm not sure if it can cause some problem later.
My question is, why will repetitive sequence cause problem during the PCR? Can repeat sequences with very short length (about 10bp) also cause the same problem?
Thanks for the reading.
I can tell you that much: I tried amplifying a some 30mer of a 12bp DNA sequence via PCR with flanking distinct primers. Even though I tried having the melting temperature of these primers above the melting temperature of my monomers, I ended up with a visible precipitation in my tube. I guess as the polymer continuously increases its melting temperature due to its length, it started to prime itself and just uncontrollably formed extensive polymers that condensed to the analogy of a long loose threat in your pocket at the end of the day.
I abandoned the project afterwards as it wasn't worth the hassle.
Short repeats of up to 20-30bp shouldn't become much of a problem during PCR (depends on the melting temperature compared to your primers), as you can both amplify and sequence His-Tag sequences which are highly repetitive. As soon as they are long enough to aneal and act as their own primer sequences, you'll get into trouble.
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