The present teachings pertain to methods, reaction mixtures, and kits for ligating polynucleotides. In some embodiments, a heat-activatable ligation agent, a phosphorylation agent, and a decontamination agent are included in the same ligation reaction mixture with at least one probe set, at least one linker set, and at least one target polynucleotide. A reaction at a first temperature results in hybridization of the probes to the target, phosphorylation of the probes, and decontamination of unwanted reaction components. A reaction at a second temperature results in the ligation of the probes together. In some embodiments, the present teachings are applied in highly multi-plexed ligation reactions in which a plurality of single nucleotide polymorphisms in a plurality of target polynucleotides are queried, and eventually detected using a mobility dependent analysis technique.