The dual ligation hybridization assay (DLA) extends the specificity of the hybridization-ligation assay to a   specific method for the parent compound. Despite hybridization-ligation assays robustness, sensitivity and added specificity for the 3-end of the oligonculeotide analyte, the hybridization-ligation assay is not specific for the 5' end of the analyte.
The DLA is intended to quantify the full-length, parent oligonucleotide compound only, with both intact 5 and 3 ends. DLA probes are ligated at the 5 and 3 ends of the analyte by the joint action of T4 DNA ligase and T4 polynucleotide kinase. The kinase phosphorylates the 5-end of the analyte and the ligase will join the capture probe to the analyte to the detection probe. The capture and detection probes in the DLA can thus be termed ligation probes. As for the hybridization-ligation assay, the DLA is specific for the parent compound because the efficiency of ligation over a bulge loop is low, and thus the DLA detects the full-length analyte with both intact 5 and 3'-ends. The DLA can also be used for the determination of individual metabolites in biological matrices.
The limitations with the hybridization-ligation assay also apply to the dual ligation assay, with the 5-end in addition to the 3-end requiring to have a free hydroxyl (or a phosphate group). Further, T4 DNA ligases are incompatible with ligation of RNA molecules as a donor (i.e. RNA at the 5 end of the ligation). Therefore, second generation antisense that comprise 2-O-methyl RNA, 2'-O-methoxyethyl or locked nucleic acids may not be compatible with the dual ligation assay.