Investigating the compatibility of polymerase chain reaction and oligonucleotide ligation assay for one step detection of point mutations

We present a systematic optimization of the components of polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to combine them into a one-pot reaction. Such a one-pot reaction would enable the simultaneous amplification of a target nucleic acid sequence via PCR (for species identification) and the detection of a point mutation within that sequence via OLA (for the determination of antimicrobial resistance). The results can be visualized directly on a lateral flow assay without additional equipment. Specifically, here, we amplify a 163 bp region of the rpoB gene of Mycobacterium tuberculosis and detect the 531 mutation therein. The concentrations of each component of PCR and OLA buffer were optimized to design a new buffer compatible with the combined reaction. An important finding of the study is that the performance of PCR and OLA is highly sensitive to magnesium ion (Mg2+) concentration, and the incompatibility in the Mg2+ concentration is preventing the integration of the two reactions in the present study. Using a DNA polymerase that can tolerate high Mg2+ concentration might lead to the successful integration of PCR and OLA. Compared to the existing methods of conducting the PCR and OLA reaction sequentially, the one-pot reaction would significantly reduce the reaction time, user intervention, and plausible risk of amplicon contamination arising from opening the PCR tubes.