1. Glycerol prevents evaporation and enhances exonuclease digestion and polymerization in concentrations of 5-20%. Such concentrations may be reached without the addition of extra glycerol if, for example, the nucleotides and the relevant buffers are stored in 50% glycerol. Storing key reagents in 50% glycerol has the advantage that they can be pipetted directly from -20°C, thus avoiding freezing and thawing. The activity of the restriction enzyme also can be enhanced with glycerol, but this enhancement may not be desirable, as it takes the form of star activity, with the enzyme cutting at relaxed specificity.

2. The criterion for the selection of these identifiers is that they should specifically recognize a given rolling-circle product—any oligonucleotide from 16-mers and up that does not recognize genomic sequences should be suited for the purpose.

3. Because it is possible to co-hybridize and co-roll multiple padlock probes on the same specimen, it is preferable to generate a class of unique identifiers that can also be co-hybridized for the identification of the individual rolling-circle products in situ. Each identifier is synthesized with one fluorophore attached to it, and the fluorophores are chosen among the fluorophores usually used for FISH. Instead of using labeled oligonucleotides and FISH to identify the rolling-circle products, the identifier oligonucleotides may also be used as PRINS primers, e.g. in a DNA cascade reaction (1). However, the FISH procedure is the best tested at this point and the approach best suited for multiplexing. For PRINS detection, it may be preferable to design the backbone from only three nucleotides so it can be detected by dideoxy PRINS.

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