Development Of DNA Chip For Mutation Detection Of Multidrug-resistant Mycobacterium Tuberculosis

The recent resurgence of tuberculosis has been accompanied by an increase in primary and acquired resistance of Mycobacterium tuberculosis (M. tuberculosis) to antibiotic agents. On the other hand, drug resistance mechanisms have been extensively investigated on the genomic basis and there are already adequate findings. For examples, it has been identified that many genetic mutations leading to rifampin, isoniazid, ethambutol, or pyrazinamide resistance. For example, most rifampin resistance in M. tuberculosis is associated with mutations in a 81 bp DNA fragment located in the rpoB gene encoding the β subunit of RNA polymerase. Thus, reliable rapid mutation detection and genotyping technologies are urgently needed to detect drug resistance.In order to provide templates and quality control in our research, some mutation gene fragements including rpoB, katG, embB, rrs, spsL, obtained by splicing by overlap extension were cloned to T-vector. Proofreading PCR (PR-PCR), which 3' end of one primer were aligned with putative mutation site and 3' hydroxyls of the primer were blocked by amino groups, was applied to detect single base mutation in the rpoB gene of M. tuberculosis by utilizing the proofreading ability of some DNA polymerases. The polymerase can distinguish the mutant gene from wild type by removing the blocked 3'terminal nucleotide when the primer is mismatch-paired with the template. Results showed that PR-PCR could be used to mutation discrimination but he polymerase proofreading activity was easily affected by some factors including ion, temperature, primer-template combination. Solid phase hybridization-ligation based on the contiguously stackinghybridization (CSH) and ligation of T4 DNA ligase was developed to detect mutations in the rpoB gene on the DNA chip. the immobilized probes and short probes were hybridized with target DNA, and then the two probes were ligated by T4 DNA ligase. The second base of 3' terminus of the short probe aligned with a putative mutation and 5' terminus was modified with biotin. In the hybridization processing, only when short probe perfectly matched with template, it can be ligated with the immobilized probe by DNA ligase. Single-base mutation and also multiplex mutations of 516, 526 and 531 sites in the rpoB gene could be identified on the DNA chip. The hybridization-ligation assay on DNA chip is useful for detection gene mutations associated with drug resistance, and even more for high-throughtout SNP assay.