Genomic dna enzymatic labelling kit

Array experiments were performed as recommended by the manufacturer (Agilent Technologies). DNAs (500 ng) from the specimen and a sex-matched reference (Promega, Madison, WI, USA) were double-digested with RsaI and AluI for 2h at 37°C. After heat inactivation of the enzymes at 65°C for 20 min, each digested sample was labelled by random priming (Genomic DNA Enzymatic Labelling Kit, Agilent) for 2 h using Cy5-dUTP for patient DNAs and Cy3-dUTP for reference DNAs. Labelled products were column-purified using Microcon Ym-30 filters (Merck Millipore Corporation, Darmstadt, Germany). Hybridization was performed at 65°C with rotation for 24 h. After two washing steps, the array was analyzed with the Agilent scanner using the Feature Extraction software (Agilent Technologies).

High-density aCGH were performed using SurePrint G3 Human CGH Microarray 1 × 1 M (Agilent), an oligonucleotide chip that contains 963,029 distinct biological features with a probe spacing 2.1 KB overall median probe spacing (1.8 KB in Refseq genes) and Content sourced from - UCSC hg18 (NCBI Build 36).
Array experiments were performed as recommended by the manufacturer (Agilent Technologies, Santa Clara, CA, USA). 500 ng of DNA from the patient and a reference sample of the same sex (Promega, Madison, WI, USA) were double-digested using AluI and RsaI for 2 h at 37 °C. Enzymes were inactivated at 65 °C for 20 minutes and each digested sample was labeled with Cy5-dUTP by random priming at 37 °C for 2 h (Genomic DNA Enzymatic Labelling Kit Agilent). Labeled products were column-purified (Microcon Ym-30 filters, Millipore Corporation). The hybridization was performed at 65 °C with rotation for 24 h after probe denaturation and pre-annealing with Cot-1 DNA. The array was analyzed with the Agilent scanner using the Feature Extraction software (v9.1 Agilent Technologies). Comprehensive description of the statistical algorithms is available in the user’s manual provided by Agilent Technologies.