Earlier serological typing approaches had been established for differentiating bacterial isolates, but immunological typing has drawbacks such as reliance on few antigenic loci and unpredictable reactivities of antibodies with different antigenic variants. Several molecular typing schemes have been proposed to determine the relatedness of pathogens such as pulsed-field gel electrophoresis (PFGE), ribotyping, and PCR-based fingerprinting. But these DNA banding-based subtyping methods do not provide meaningful evolutionary analyses. Despite PFGE being considered by many researchers as the “gold standard”, many strains are not typable by this technique due to the degradation of the DNA during the process (gel smears).
The approach of MLST is distinct from Multi locus enzyme electrophoresis (MLEE), which is based on different electrophoretic mobilities (EM) of multiple core metabolic enzymes. The alleles at each locus define the EM of their products, as different amino acid sequences between enzymes result in different mobilities and distinct bands when run on a gel. The relatedness of isolates can then be visualized with a dendrogram generated from the matrix of pairwise differences between the electrophoretic types. This method has a lower resolution than MLST for several reasons, all arising from the fact that enzymatic phenotype diversity is merely a proxy for DNA sequence diversity. First, enzymes may have different amino acid sequences without having sufficiently different EM to give distinct bands. Second, "silent mutations" may alter the DNA sequence of a gene without altering the encoded amino acids. Thirdly, the phenotype of the enzyme can easily be altered in response to environmental conditions and badly affect the reproducibility of MLEE results - common modifications of enzymes are phosphorylation, cofactor binding and cleavage of transport sequences. This also limits comparability of MLEE data obtained by different laboratories, whereas MLST provides portable and comparable DNA sequence data and has great potential for automation and standardization.
MLST should not be confused with DNA barcoding. The latter is a taxonomic method that uses short genetic markers to recognize particular species of eukaryotes. It is based on the fact that mitochondrial DNA (mtDNA) or some parts of the ribosomal DNA cistron have relatively fast mutation rates, which give significant variation in sequences between species. mtDNA methods are only possible in eukaryotes (as prokaryotes lack mitochondria), whereas MLST, although initially developed for prokaryotes, is now finding application in eukaryotes and in principle could be applied to any kingdom.