Theoretical and Experimental DNA Computation (Natural ...

5.9 Other Laboratory Implementations 135
far lower than that of the S2 and S3 primers, we observed incomplete removal
of S1 sequences. As a result of these problems, we redesigned the strands, the
modifications being detailed in [6].
PCR can introduce problems
It is unrealistic to assume that our enzymatic method removes 100% of the
targeted strands. We must therefore be prepared to accept that a small proportion
of target strands will be left in solution. Normally, this residue would
be undetectable, but the repeated use of PCR can quickly amplify this trace
amount, causing failure of the algorithm being implemented. The experiment
confirmed that our removal method worked, but the use of PCR as a detection
method was far too sensitive for our purposes. Kaplan et al. [87] confirm our
belief that PCR is a major source of errors.
Biotinylated strands can introduce problems
In our experiments we used a biotinylated primer to purify away the “splint”
strands used to construct the initial library. Quite apart from the problems
with biotinylation described earlier, it became clear from our investigations
that this can cause other significant difficulties. We found that the attached
beads “settled out” in solution, dragging the strands to the bottom of the
heating block and affecting the efficiency of the process. We overcame this
problem by incubating the tubes in a rotating oven.
Restriction enzymes are often not as effective as they are claimed
to be
Although various claims are made for the efficiency of restriction enzymes,
in reality they have a nonzero error rate associated with them. We found
that Sau3A was ineffective at cleaving double-stranded DNA, but that MboI
worked perfectly well. This may have been due to the fact that Sau3A is
inefficient at cleaving de novo synthesized DNA.
5.9 Other Laboratory Implementations
In this section we describe several successful laboratory implementations of
molecular-based solutions to NP-complete problems. The objective is not to
give an exhaustive description of each experiment, but to give a high-level
treatment of the general methodology, so that the reader may approach with
confidence the fuller description in the literature.