Theoretical and Experimental DNA Computation (Natural ...

Experiment 25. New full-length chain preparation
5.8 Experimental Investigations 129
Three new full-length chains were prepared containing either red, green, or
blue new v3 in a backbone of the original red tiles v1, v2, v4, v7, andv8.
By omitting the green and blue tiles in the backbone of the molecule we
hoped to avoid any interactions between blue CCCCCC and green GGGGGG
sequences, which could lead to the formation of hairpins in the full-length
molecule. The construction method was exactly as in Experiment 2.
Experiments 26 to 29. New chain amplification
Each of the new chains were amplified and purified separately to produce
both biotinylated and non-biotinylated products. The non-biotinylated products
were cloned for sequencing (but unfortunately the sequencing reactions
failed and there was not enough time to repeat them). The biotinylated products
were used in the control and exclusion optimization experiments.
Experiments 30 and 31. PCR control experiments for the new tile
chains
The three new tile chains containing either the red, green, or blue v3 were
assessed separately. Serial dilutions were made from each template (down to
a dilution of 10:8). PCR reactions were then set up in order to determine
the limit of detection for each template. For subsequent control and exclusion
reactions these templates were used at a concentration approximately tenfold
above the limit of detection. It was hoped that by balancing the initial
amount of template used in the experiments, any partial exclusion (as seen
previously) would be sufficient to reduce the level of template below the limit
of PCR detection, giving a negative result (i.e., showing that exclusion had
been successful). PCR conditions were optimized to ensure that there was no
cross reaction between the new colored sequences, and Mbo1 digestion times
were assessed to ensure complete digestion of double-stranded DNA at these
concentrations.
Experiment 32. Final exclusion experiment
Red, green, and blue templates were mixed in roughly equal proportions
(based on their PCR detection limit in Experiment 30). The mixed template
was bound to the Dynabeads and prepared in bulk before splitting into four
tubes. Red, green, and blue v3 exclusions were set up in separate reactions,
alongside a no-exclusion control. Following Mbob digestion, detection PCR
reactions were set up to determine the relative levels of each v3 in each of the
exclusion samples and control.