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Mathur M.,Molecular Biology Division | Singh E.,Bhabha Atomic Research Center | Poduval T.B.,Bhabha Atomic Research Center | Rao A.V.S.S.N.,Molecular Biology Division
Indian Journal of Medical Research

Background & objectives: Wilson’s disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson’s disease. Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson’s disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis. Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations. Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders. © 2015, Indian Council of Medical Research. All rights reserved. Source

Agarwal R.,Molecular Biology Division | Maralihalli G.,Molecular Biology Division | Sudarsan V.,Chemistry Division | Choudhury S.D.,Bhabha Atomic Research Center | And 4 more authors.
Journal of Bioenergetics and Biomembranes

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40 k segments), 90,000×g (90 k segments) and 150,000×g (150 k segments). Electron microscopy showed differences in their appearance. 40 k segments looked feathery and fluffy, whereas the 90 k and 150 k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40 k, 90 k and 150 k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77 K (λex0435 nm) was highest in 150 k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ10<40 ps, τ20425-900 ps and τ302.4-3.2 ns. The percentage contribution of the fastest component (τ1) decreased in the order 40 k>90 k>150 k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803. © 2012 Springer Science+Business Media, LLC. Source

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