Theoretical and Experimental DNA Computation (Natural ...

5.8 Experimental Investigations 127
This sample was bound to the Dynabeads, washed but not denatured, and
then taken through the procedure as double-stranded template, which should
have been completely destroyed by the exclusion procedure.
Experiment 13. Sau3A control
This control was used to assess the ability of Sau3A to digest double-stranded
DNA. Serial dilutions were made from template ALL. 1 µl of each dilution
was digested using 10U Sau3A at 37 ◦ C for one hour, in a total volume of
10 µl. 1 µl of each digest, and undigested controls, were used as template in
standard v1–v8 detection PCR reactions.
Experiments 14 and 15. Control experiments using Mbo1 to digest
targeted sequences
Experiment 13 was repeated using Mbo1(aSau3A isoschizomer that has the
same specificity for double stranded DNA).
Experiment 16. Exclusion experiment 4
Experiment 12 was repeated, substituting Mbo1forSau3A.
Experiment 17. Exclusion experiment 5
Fresh v1–v8 template was prepared and purified, then used in an exclusion
experiment as in Experiment 12. Digestion was carried out overnight using
50U enzyme.
Experiment 18. Exclusion experiment 6
Template prepared in Experiment 17 was diluted 1/10 and 1 µl bound to
Dynabeads, denatured, and washed. A v2 exclusion was set up, using Taq in
the primer extension reaction as usual. The beads were re-suspended every 10
cycles and a 3 µl aliquot of beads removed at roughly 20 cycle intervals, up
to a maximum of 85 cycles. Mbo1 digestion of each aliquot was carried out
overnight at 37 ◦ C in a rotating oven (to keep the beads in suspension) using
50U (a huge excess) of enzyme. v2 detection PCRs were set up from each of
the digested samples.
Experiment 19. Exclusion experiment using Klenow
In this experiment Klenow was substituted for Taq in the primer extension
step. The idea was to overcome the problem of the beads settling out while
cycling in the PCR block, which may have accounted for the inefficiency of
the reaction. To do this the tubes were incubated in a 37 ◦ C rotating oven for