Theoretical and Experimental DNA Computation (Natural ...

130 5 Physical Implementations
Results obtained
Experiment 3. The major product of ∼200 b.p. was detectable at all template
concentrations, though very faint at 1/1000 dilution. MgCl2 concentration
had very little effect on PCR efficiency. Optimal conditions appeared to
be at 2mM MgCl2 using the template diluted to 1/10. In all cases the product
appeared slightly smaller than expected, though it was not clear if this was a
gel artifact or a problem with the library oligo assembly.
Experiment 5. All three vertices were found to be represented in the chain
population (assuming no cross reaction had occurred)
Experiment 6. The PCR products were faint but in each case appeared to
be color-specific. There was also a stepwise reduction in the size of the PCR
product from v1 through v4, showing that the PCR reactions were vertexspecific
and that the majority of colorings had assembled in the correct order.
Experiments 7 and 8. It was clear that there was fairly high sequence variability
among the clones. They showed a number of vertex assembly patterns
and chain lengths. Some could be explained by PCR mispriming during the
chain amplification step, yielding products with a v1 sequence at both ends
(these products would not cause problems in the exclusion experiments since
they were not biotinylated, would not bind to the Dynabeads, and would
be removed during the washing step). One feature common to all the clones
was the absence of sequences representing v5 and v6. Looking at the oligo sequences
it was clear that the problem was due to identical overlapping regions
between v4 and v5, andv6 and v7, making two chains possible, the shorter
of which seemed to form predominantly. In this small selection of clones it
looked like the vertex colorings were represented equally among the chains.
Experiment 9. The detection reactions showed that the PCR products from
each sample were of equal intensity for each vertex tested, showing that exclusion
under these conditions had failed.
Experiment 11. The PCR results showed that the detection step was specific,
but that the exclusion steps had not reduced the amount of targeted
sequence.
Experiment 12. Detection PCR results showed that the PCR was specific
(RED control), but that the exclusion steps had not worked, and also that
Sau3A had failed to destroy the double-stranded control. This implied that
the exclusion experiments were failing due to incomplete Sau3A digestion of
the marked sequence.