Theoretical and Experimental DNA Computation (Natural ...

126 5 Physical Implementations
Experiment 6. PCR from RED template (from Experiment 2)
This control experiment was set up to check the specificity of the PCR detection
step (to make sure the color-specific primers did not cross-react, giving
false positive results). The RED hybridization product from Experiment 2
was diluted 1/10 and color-specific detection PCRs set up for v1, v2, v3, and
v4.
Experiments 7 and 8. Sequencing of clones from Experiment 4
Eight clones were sequenced using either universal or reverse primers.
Experiment 9. Exclusion experiment 1
This initial control experiment was designed to test the ability of the proposed
Taq-based primer extension/Sau3A digestion method of excluding specific sequences
from a mixed population of chains. Template prepared in Experiment
4 was bound to Dynabeads, denatured and then split into two. One half was
treated with the intention of excluding all but one vertex coloring, the other
was used as a control and taken through the procedure without adding any
exclusion primers. Following Sau3A digestion, the surviving DNA was harvested
from the beads by EcoR1 digestion. Detection PCR reactions were set
up to detect specific vertex sequences within each of the samples. The untreated
half was intended as a positive control for the detection PCR step.
Experiment 10. Amplification and purification of the RED template
A red-only template was prepared by PCR from the RED hybridization product
(Experiment 2) using v1 = red and v8 primers. The PCR product was gel
purified and extracted using a Qiagen gel extraction kit.
Experiment 11. Exclusion experiment 2
This was a repeat of Experiment 9 with various modifications intended to
increase the stringency of the exclusion step (amount of template reduced,
primer concentration increased, annealing temperature reduced, number of
cycles increased). The RED control template was also taken through the procedure
as a PCR specificity control.
Experiment 12. Exclusion experiment 3
Conditions were modified further to favor exclusion. An additional control
was included to test the ability of the Sau3A to destroy double-stranded
sequences.