Slides were then rinsed in PBS, twice in double distilled water and then dehydrated through ethanol series as before. Nuclei on the slide were digested with pepsin (100 μg/ml) in 0.01 N HCl for 20 min at 37☌, rinsed in double distilled water and followed by PBS, and fixed in 1 per cent paraformaldehyde in PBS for 10 min at 4☌. Slide was washed in acetic acid for two min, and dehydrated in 70, 90, 100 per cent ethanol, three min each. Working concentration of probe DNA was between 100-200 ng/μl. All probes were labelled using nick translation method 7 with FITC-12-dUTP (Roche, Germany) or TRITC-12-dUTP (Roche, Germany) or C圓 (Amersham, UK). Probe DNA was extracted using a commercial BAC extraction kit (Sigma, India). The clones were received as bacterial (LB) agar stab culture, which were sub-cultured on LB agar plate before growing in large amount in LB medium. Bacterial artificial chromosome (BAC)/Phage artificial chromosome (PAC) clones ( Table II) were obtained from European Resource Centre for Molecular Cytogenetics, University of Bari, Italy ( Courtesy: Prof. Microdeletion status was determined by FISH using non-commercial DNA probes. Metaphase spreads were prepared from phytohaemagglutinin stimulated human peripheral blood lymphocytes using standard cytogenetic technique 7. Approximately 20 μl of cell suspension was used to prepare the slide. Cells were finally re-suspended in 100 μl of fresh fixative. In brief, blood nucleated cells were washed in phosphate buffer saline (PBS), three times before hypotonic treatment (50 mMol KCL) for 30 min and fixation in methanol:acetic acid solution (3:1). Interphase cell suspension was prepared by standard method 7. This study was aimed to assess the role of FISH for the rapid detection of various clinically suspected microdeletion syndromes. Thus, FISH provides a high-resolution analysis of only targeted locations. However, conventional FISH does not allow a comprehensive evaluation of the whole genome. The ability of FISH to detect cryptic chromosomal rearrangements exceeds the resolution of any form of cytogenetic banding techniques. These limitations of conventional chromosome analysis have been overcome by FISH 5, 6. In addition, most rearrangements of the ends of the chromosomes (telomere or sub-telomere) are too small to be detected using traditional banding technique. However, despite its indisputable success, this tool has limited resolution, usually being unable to detect genomic changes of less than 5 Mb 3, 4. G-banded karyotyping is the most common approach for the detection of genomic alterations. The known microdeletion syndromes are DiGeorge/Velocardiofacial (22q11.2), Prader-Willi/Angelman (15q11-13), William (7q11.23), Smith-Magenis (17p11.2), Cri-du-Chat (5p15.2), Miller-Dieker (17p13.3), WAGR (Wilms tumour, Aniridia, Genitourinary anomalies and mental Retardation 11p13), HNPP (Hereditary Neuropathy with Liability to Pressure Palsy 17p12), Wolff Hirschhorn (4p16.3), TRPS (Tricho-Rhino-Phalangeal 8q24.1), ATR 16 (Alpha Thalassaemia mental Retardation-16 16p13.3), etc. This is frequently associated with multiple congenital anomalies and developmental delay 3, 4. It is mostly spontaneous, and is reported to occur in approximately 5 per cent of patients with unexplained mental retardation 1, 2. The microdeletion syndrome is characterized by hemizygous microdeletion (<5 MB) of chromosomes in which one or more genes are lost.
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