3.1. Design of starting components for LASSO probe synthesis
The major innovation in this new method of LASSO production was the introduction of a plasmid-mediated process to amplify and reconfigure a LASSO probe library with improved control to minimize unwanted byproducts of probe self-circularization. The assembly procedure we developed begins with the two main components: a precursor probe (pre-LASSO probe, Fig. 1a .) that is a ssDNA oligonucleotide (158bp) which contains the ligation and extension arms designed to hybridize with sequences that flank the targeted region and a plasmid vector which we refer to as pLASSO (Fig. 1b. ). The role of the pLASSO plasmid is to supply the backbone (in blue) for a mature LASSO (Fig 1c .) and a number of functional sites required for the assembly of a mature LASSO. The pLASSO vector was obtained in house starting from the pLox2+ linear plasmid (NEB) and a synthetic DNA fragment (backbone) as described in the material and methods. Uniquely, this plasmid was customized to have two LoxP sites (purple triangles) for Cre-recombination and a linearization primer annealing site for linearization. The plasmid also contained an ampicillin-resistance gene for selection in E.coli. In order to start the assembly of the LASSO probe, pLASSO needs to be linearized by PCR with linearization primers that have tails a and b identical to the a and b ends (primer selector annealing sites) of the pre-LASSO probe.