I require large amounts of small (16bp) dsDNA molecules for zinc finger binding studies. This is the major bottleneck in my research since the large amounts are expensive to purchase synthesized even in large scale and I accordingly perform fewer experiments than I'd like.
I have been looking into ways to express these DNA molecules in a bacterial system with reasonable yield and I am currently looking into the possibility of engineering retrons in order to produce custom msDNAs that I could then digest with restriction enzymes and purify. Would it be possible to replace the sequence corresponding to the complementary portion of the DNA strand with both strands of my DNA sequences, flanked by restriction sites? I imagine placing my engineered operon in an expression plasmid would enable me to induce the production of large amounts of msDNA with IPTG.
Wait a minute! msDNA is RNA-DNA hybrid plus protein complex... Thus, I do not understand the whole rationale behind this idea.
How much dsDNA do you need?
could you design an oligo with your sequence in the middle and blunt end cutting restriction sites at each end of your sequence of interest and either clone this and cut it out of the vector or include short primer binding sequences at each end of your sequence and pcr, digest and page purify your product. seems a little more direct but it depends on quite how much product you need
Just to follow up (and also for my own records), my idea seems somewhat plausible given the paper cited below, however since I would be expressing msDNA with minimal mismatches the yield would be fairly low (native sequences tend to give better yields and have mismatches). Estimating conservatively, I would need 2L of culture for one experiment.
In answer to Olga Novikova's question: yes, it's single-stranded, but it bends back to base-pair with itself. If there are restriction sites flanking a paired sequence, then you can generate double-stranded DNA from the msDNA.
http://www.rle.mit.edu/sbg/wp-content/uploads/2014/11/Genomically-encoded-analog-memory-with-precise-in-vivo-DNA-writing-in-living-cell-populations-Combined.pdf
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