The HDPE incorporation in the first stage not only resolves the reaction engineering problems, but also contributes to the higher isotactic stereoregularity of 1-butene polymerization.
The expected signal is (0,0,0,1)—no incorporation in the first three flows, and an incorporation of a single nucleotide in the fourth flow.
Therefore, there are three possible candidates for the next nucleotide A, C and T. Next, assume a different scenario an incorporation event happened only in the second flow, with no incorporation in the first and third flows.
In this case, the sequence starts with C, and the second nucleotide cannot be G (otherwise we would have seen an incorporation in the third flow), and it cannot be C (otherwise we would have seen two incorporations in the second flow).
Hence, there are only two candidates for the next nucleotide A and T. In the first scenario, the previous probability of observing an incorporation in the fourth flow is, as T is one of three possible nucleotides, whereas in the second scenario, the previous probability is, as T is one of only two possible nucleotides.
These data can also be used to guide their incorporation into the first-in-human studies, a major goal in phase 0 trials for molecular proof of concept [ 14].
Assume, for example, the same flow order as before, and assume that we have seen incorporations in the first three flows.
This implies that the sequence starts with ACG, and that the next base in the sequence is not G (otherwise we would have seen two incorporation events in the third flow).
For example, in the intermediate ABA, A1 has been incorporated in the first branch, A2 and B2 are in the second branch, and A3 is in the third branch (note again that A2 incorporation is a prerequisite of B2 incorporation).
Then double-stranded (ds) cDNA synthesis was performed with the incorporation of dUTP in the second strand.
