text: In the hybridization-ligation assay a template probe replaces the capture probe in the sandwich assay for immobilization to the solid support. The template probe is fully complementary to the oligonucleotide analyte and is intended to serve as a substrate for T4 DNA ligase-mediated ligation. The template probe has  in addition  an additional stretch complementary to a ligation probe so that the ligation probe will ligate onto the 3'-end of the analyte. Albeit generic, the ligation probe is similar to a detection probe in that it is labelled with, for example, digoxigenin for downstream signalling. Stringent, low/no salt wash will remove un-ligated products.
  The ligation of the analyte to the ligation probe makes the method specific for the 3-end of the analyte, ligation by T4 DNA ligase being much less efficient over a bulge loop, which would happen for a 3 metabolite N-1 version of the analyte, for example. The specificity of the hybridization-ligation assay for ligation at the 3-end is particularly relevant because the predominant nucleases in blood are 3 to 5' exonucleases.
  One limitation of the method is that it requires a free 3-end hydroxyl which may not be available when targeting moieties are attached to the 3-end, for example. Further, more exotic nucleic acid chemistries with oligonucleotide drugs may impact upon the activity of the ligase, which  needs to be determined on a case-by-case basis.