Characterization of a Trinucleotide Repeat Sequence (CGG) ₅ and Potential Use in Restriction Fragment Length Polymorphism Typing of Mycobacterium tuberculosis

Abstract

The genomes of 28 bacterial strains, including mycobacterial species Mycobacterium tuberculosis and Mycobacterium bovis, were analyzed for the presence of a special class of microsatellite, that of trinucleotide repeat sequences (TRS). Results of a search of all 10 possible TRS motifs (i.e., CCT, CGG, CTG, GAA, GAT, GTA, GTC, GTG, GTT, and TAT) with five or more repeating units showed that (CGG) 5 DNA fingerprinting of the inserted IS6110 element specific for the Mycobacterium tuberculosis complex is a powerful epidemiological tool for visualizing DNA restriction fragment length polymorphisms (RFLP) of M. tuberculosis (26). The major limitation of IS6110-based RFLP typing is the difficulty of discriminating genetic polymorphisms of M. tuberculosis isolates with only a few copies of the element. In addition, there are two reports (1, 29) that described IS6110-based RFLP as unstable, although other studies have confirmed a high degree of stability (5, 15). Yeh and colleagues (29) indicated that genotypes with IS6110 were relatively unstable because they changed rapidly compared with those based on another marker. Alito et al. (1) reported that a multidrugresistant outbreak strain changed rapidly, according to IS6110 RFLP, over a period of a few years.

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

“…Therefore, (CGG) 5 -based fingerprinting was suggested to be useful for typing of M. tuberculosis strains with no or only a few IS6110 copies or as a method complementing IS6110-RFLP analysis. The (CGG) 5 repeat sequences were also identified in NTM species, including M. marinum, M. kansasii, M. szulgai, suggesting that (CGG) 5 -based typing could be extended beyond MTBC strains (393). Recently, single-primer PCR targeting the TRS within the M. gordonae genome was proposed.…”

“…All bacterial genomes carry various numbers of trinucleotide repeat sequences (TRSs), which can be useful for genotyping purposes. Most of the TRSs in the genomes of mycobacteria have been localized within the genes of the PE and PPE families (393). A subgroup of the PE proteins, whose N termini harbor characteristic Pro-Glu (PE) motifs, contains polymorphic GC-rich sequences (PGRSs), just as a subgroup of the above-discussed PPE proteins contains MPTRs (394).…”

Development and Evaluation of a Line Probe Assay for Rapid Identification of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Strains

“…Two different methods were applied to extract genomic DNA. One method was described previously (12). The other method was performed as follows.…”

(CGG) 4 -Based PCR as a Novel Tool for Discrimination of Uropathogenic Escherichia coli Strains: Comparison with Enterobacterial Repetitive Intergenic Consensus-PCR

“…Since the different trinucleotide repeat sequence elements vary in copy number and distribution in bacterial genomes, they have the potential to serve as valuable markers for phylogenetic and epidemiological studies. A (CGG) 5 hybridization probe has been successfully used in conjunction with restriction fragment length polymorphism to type Mycobacterium tuberculosis (28). However, such hybridization techniques require the isolation of large amounts of genomic DNA and are time-consuming and expensive.…”