WHOLE GENOME ANALYSIS BY ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION IN PATIENTS WITH CONGENITAL MALFORMATIONS
Dimova I1, Vazharova R1, Nikolova D1, Tincheva R2, Nesheva D1, Uzunova Y3, Toncheva D1,*
*Corresponding Author: Professor Draga Toncheva, Department of Medical Genetics, Medical University Sofia, 2, Zdrave str., 1431 Sofia, Bulgaria; Tel./Fax: +359-2-952-0357; E-mail:dragatoncheva@yahoo.com
page: 33

MATERIALS AND METHODS

 

The study was approved by the Ethics Committee of the Medical University of Sofia, Sofia, Bulgaria. The parents of the children provided their informed consent. Peripheral blood was taken for genetic diagnostic testing. Chromosome Analysis. G-banded chromosomes were prepared from whole blood samples using standard laboratory protocol. DNA Extraction. DNA was extracted from blood samples by phenol-chloroform. The yield and purity for protein/RNA were estimated by a Nanodrop 1000 spectro photometer (Thermo Scientific, Wilmington, DE, USA). The DNA was checked on a 1% agarose gel: DNA of high molecular weight (>50 kb) indicated it was suitable for use. Genomic Arrays. We have used genomic array Cyto Chip (BlueGnome, Cambridge, Cambridgeshire, UK), cov ering the entire genome by bacterial artificial chromosome (BAC) clones at a median 565 kb, a resolution optimized to detect pathogenic imbalances while minimizing polymorphisms. In addition, sub-telomeric clones were included at a median 250 kb resolution, thus allowing reliable detection of mosaicism. The BAC clones of 90 known genetic conditions at a median 100 kb resolution were also included in the chip. This resulted in an average density of 1 clone/0.5 Mb. Probe Labeling, Hybridization, Image Capture and Data Analysis. Test- and sex-matched reference genomic DNA (400 ng) was labeled by random-priming, using a fluorescent labeling system (BlueGnome). The labeled products were puri- fied by AutoSeq G50 columns (Amersham Pharmacia Biotech Inc., Piscataway, NY, USA), and the incorporation of dyes was evaluated by the spectrophotometer (Nanodrop 1000, Thermo Scientific). Incorporation in the range of 6-15 pmol/L and a DNA yield of 180-325 ng/L were considered suitable for further analysis. A mix of Cy5- and Cy3- labeled probes and a mix of COT-1 and Herring sperm DNA were ethanol-precipitated at 80C for at least 30 min. Hybridization was done using dissolved precipitated probes in a hybridization buffer.Arrays were washed in sodium chloride-sodium citrate (SSC) buffers with decreasing concentrations and scanned by a GenPix 4100A (Axon Instruments, Union City, CA, USA). The images were analyzed by BlueFuse for Microarrays 3.5 software (BlueGnome). In data processing log2 ratios of Cy3 and Cy5, intensities were generated for all hybridized clones. Normal copy numbers were considered to be present if the log2 ratio was between 0.3 and +0.3, values above +0.3 were interpreted as gain/ amplification and those under 0.3 as losses (deletions). Genomic profiles were represented plotting log2 ratios in Y-axis and the 23 chromosomes in Xaxis. Individual chromosomal profiles were represented with clone positions in Y-axis and log2 ratios in X-axis.




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