Theoretical and Experimental DNA Computation (Natural ...

122 5 Physical Implementations
since the process is performed only once, rather than repeatedly during the
main body of a computation.
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V 1 V 2 V 3 V 4 V 5 V 6 V 7 V 8
Fig. 5.10. Structure of strands in initial library
We then pour all oligos into solution. When all oligos have annealed we expect
to obtain many double strands of the form depicted in Fig. 5.9. Vertex
and color sections each occupy a distinct subsection of each strand. It is clear
from the structure of these strands that the sequence encoding a particular
vertex/color combination depends not only on the vertex in question, but on
whether or not the vertex number is odd or even. For example, sections coloring
v1 “red” have the sequence AAAAAA, as expected. However, due to
the overlapping nature of the strand construction technique, sections coloring
v2 have the sequence TTTTTT. This minor complication does not present a
problem and, knowing the sequence assigned to each vertex/color combination,
it is a trivial task to derive the sequences of the appropriate primer. The
primer sequences are listed in Table 5.2.
Materials and methods
We now describe in detail the 32 experiments carried out during this particular
phase of the project. A summary of these is given in Table 5.3. The results
of these experiments are listed in the next section; here we describe only the
materials and methods used. We represent the order of experimental execution
in Fig. 5.11. Each process box is labelled with the numbers of the experiments
carried out at that stage. Note that the only cycle in the flowchart occurs
while attempting to remove strands containing a certain sequence. This is
necessitated by the need for control and optimization experiments in order to
establish optimal (or near-optimal) experimental conditions.
Experiment 1. Oligos and reagents
All the tile oligos were resuspended to 100 pmoles/µl in distilled water, then
diluted to produce two mastermixes, the first containing all the oligos and the
other containing only red oligos. The final concentration in each case was 2.5
pmoles each oligo/µl. The primer oligos were re-suspended to 100 pmoles/µl
stocks and also diluted to 30 pmoles/µl PCRworkingstock.Thebiotinylated
primer (v8) was re-suspended to 200 pmoles/µl and stored in 20 µl singleuse
aliquots. All oligos were stored at -20 ◦ C. A 5xPolymerase/Ligase buffer was
made up (based on the requirements for second strand cDNA synthesis).