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.