Theoretical and Experimental DNA Computation (Natural ...

5.8 Experimental Investigations 131
Experiment 13. Comparisons of the two sets of samples showed that digestion
with Sau3A made no difference to the detection limit of the PCR
reactions. As a further control, the products of the above reactions were gel
purified and split in two. One half was digested with Sau3A and visualized
alongside the other (undigested) control on 2% agarose. The undigested DNA
ran as a distinct band, whereas the digested half appeared as a high molecular
weight smear.
Experiments 14 and 15. An overnight 37 ◦ C digestion using 20U of enzyme
was found to completely destroy the template (i.e., to reduce the level of template
below the limit of PCR detection).
Experiment 16. Nothing could be concluded from this set of reactions since
the positive PCR controls failed.
Experiment 17. The positive and negative detection PCR controls worked,
but all other reactions failed. The problem seemed to be due to inefficient
harvesting of the excluded template from the Dynabeads prior to detection
PCR. To get round this problem an unbiotinylated v8 primer was ordered.
Using this primer, detection PCRs could be set up directly from templates
bound to the Dynabeads.
Experiment 18. The results of this experiment gave the first evidence that
the exclusion method could work. The intensity of the specific PCR product
band decreased with increased number of exclusion cycles, although the exclusion
never reached completion. The template was still detectable after 85
cycles of primer extension.
Experiment 19. The use of Klenow produced the same effect as ∼30 to
40 cycles of Taq-based exclusion (i.e., exclusion was not complete), showing
that Klenow offered no advantage over Taq. Detection PCR showed specific
exclusion of v2 = green and v2 = blue sequences, but not v2 = red. Thegel
is depicted in Fig. 5.12. A summarized interpretation of this gel is presented
in Table 5.4.
Lanes 1 to 3 show the result of the removal of strands encoding v2 = red. Lane
1, corresponding to v2 = red should be empty, but a faint band is visible. Lanes
2 and 3, corresponding to v2 = green and v2 = blue primers respectively contain
normal length product, showing that strands not containing the sequence
v2 = red were not removed.
We believe that the incomplete removal of v2 = red strands is due to the
sequence chosen to represent red (AAAAAA). Because adenine only forms
two hydrogen bonds with thymine, the optimum annealing temperature between
strands and red primers is lower than that for green (CCCCCC) and
blue (GGGGGG) primers. We believe a simple modification to the encoding
sequence (described later) will solve this problem.