Capillary Electrophoretic Restriction Fragment Length Polymorphism Patterns for the Mycobacterial hsp65 Gene

Abstract

ABSTRACT PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of Mycobacterium species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein ( hsp65 ) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12 Mycobacterium species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify Mycobacterium species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of Mycobacterium species that is feasible for automation and routine use without the need for costly probes.