Temperature-Mediated Heteroduplex Analysis Performed by Using Denaturing High-Performance Liquid Chromatography To Identify Sequence Polymorphisms in Mycobacterium tuberculosis Complex Organisms

PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were oxyR, in which a specific polymorphism (G(1031)A) is found only in certain species of the Mycobacterium tuberculosis complex, and five genes in which mutations associated with antituberculosis drug resistance have been found. The resistance genes (with affected drug and PCR product sizes given parenthetically) were rpoB (rifampin; 258 bp), katG (isoniazid; 205 bp), pncA (pyrazinamide; 579 bp); rpsL (streptomycin; 196 bp), and embB (ethambutol; 185 bp). Elution patterns of heteroduplexes of all 20 polymorphisms studied shifted detectably at column temperatures ranging from 65.3 to 68°C and elution times of 3.5 to 6 min. These results show that temperature-mediated heteroduplex analysis is a potentially useful genotypic screen for mutations associated with antituberculosis drug resistance and for the G(1031)A polymorphism in oxyR. The method may allow users to detect novel as well as heterogeneous mutations without using expensive kits or detection labels.