The J-probe's simple but elegant design offers a number of key functional advantages:
The J-probe is not susceptible to "primer-dimer" reactions and other sources of spurious information. So J-probes can be used in very high concentrations, meaning that even rare or unique target sequences will reliably hybridize. Also, the reaction naturally produces many signal sequences for each target sequence. Therefore, the sensitivity of the reaction will at least match and in most cases greatly exceed that of other assays.
The target-specific hybridization of the J-probe creates a rigorous test to uniquely identify the target. In addition, the circular DNA reaction products can be "cleansed" with exonuclease to eliminate all large molecules other than the completed probe. This is a quality control step missing in almost all other assays, resulting in the highest available diagnostic specificity.
Since the molecular structure of J-probes virtually eliminates the possibility of spurious interaction among probes, the reaction can match or exceed the multiplicity of "padlock probes" (which have been tested to greater than 14,000 targets) – but without the risk of self-ligation or the restriction to a single ligated probe per target. The J-probe reaction is the only detection chemistry which can simultaneously achieve very high multiplicity and uncompromised accuracy.
Many multiplex assays require splitting samples into separate portions, either in "cartridges" or microfluidically distributed wells. This approach is inherently inferior to a single-tube reaction. Costs of separate cartridges, separate wells, or microcell chips are high; microfluidics require delicate fluid handling and precise conditions; and partitioning analytes into smaller samples risks missing less common targets. The single-tube J-probe reaction has none of these drawbacks.
No new equipment.
Some highly multiplexed assays require laboratories to invest in new equipment, from expensive mass spectrometers to custom-built single-purpose machines. The J-probe assay requires no such investment. Completed J-probes can readily be cleaved into standard single-stranded DNA and submitted to any standard detection platform, including arrays or beads.