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Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

To establish a method for the detection of isoniazid resistance-associated mutations in katG gene and inhA gene in Mycobacterium tuberculosis with single-stranded conformation polymorphism (SSCP) analysis by the micro-channel electrophoresis chip system.The polymer solutions of acrylamide and its derivatives were used for sieving matrices with small amount of acridine orange as the fluorescent tag. A novel pair of primers was designed to link an extra segment that could be base paired with the mutation region to the PCR products of wild type inhA gene, which made the single strands of wild type inhA fragments present a unique conformation.The wild type fragments of katG gene and the fragments with mutation at codon 315 can be distinguished, and the inhA fragments with wild type and mutated regulatory sequence can be distinguished by this method. Twenty-two out of the 23 resistant strains were detected from 30 clinical isolates, the efficiency being 95%.It is demonstrated the high speed and sensitivity in detecting the mutations of isoniazid-resistant genes in Mycobacterium tuberculosis by micro-channel electrophoresis, and this method may be applicable in clinical detection of isoniazid-resistant strains.