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Untersuchte Arbeit:
Seite: 23, Zeilen: 1 ff. (komplett)
Quelle: Papa 2010
Seite(n): 28 f., Zeilen: 28: 1 ff.; 29: 1 ff.
3) MATERIALS & METHODS

3.1 Patients collection

Patients with ID and MCA enrolled in this study have been selected among those referred the Medical Genetics Unit of the University Hospital of Siena. All patients were evaluated by clinical geneticists.

3.2 Array-based CGH

3.2.1 Samples preparation

Genomic DNA of normal controls was obtained from Promega. Genomic DNAs were extracted from peripheral blood samples using a QIAamp DNA Blood Maxi kit according to the manufacturer protocol (Qiagen, www.qiagen.com). The OD260/280 method on a photometer was employed to determine the appropriate DNA concentration (Sambrook 1989). Patient and control DNA samples were sonicated to produce a homogeneous smear DNA extending from approximately 600 bp to 2 Kb. DNA samples were then purified using the DNA Clean and Concentrator kit (Zymo Research, Orange, CA). Ten micrograms of genomic DNA both from the patient and from the control were sonicated. Test and reference DNA samples were subsequently purify using dedicated columns (DNA Clean and Concentrator, Zymo research, CA92867-4619, USA) and the appropriate DNA concentrations were determine by a DyNA Quant™ 200 Fluorometer (GE Healthcare).

3.2.2 Human oligonucleotides array

Array based CGH analysis was performed using commercially available oligonucleotide microarrays containing about 43,000 60-mer probes with an estimated average resolution of about 100-130 Kb (Human Genome CGH Microarray 44B Kit, Agilent Technologies) and microarrays containing 99,000 60- mer probes with an estimate average resolution of 50-65 Kb (Human Genome CGH [Microarray 105A Kit, Agilent Technologies).]


67. Sambrook, J. & M.J. Gething, Protein structure. Chaperones, paperones. Nature, 1989. 342(6247): p. 224-5.

3. MATERIALS & METHODS


3.1 Patients collection Patients with ID and MCA enrolled in this study have been selected among those attending the Medical Genetics Unit of the University Hospital of Siena. All they were evaluate in genetic counseling and a clinically recognizable condition was excluded a diagnosis of a recognizable syndrome, and all patients.

3.2 Array-based CGH

3.2.1 Samples preparation

Genomic DNA of normal controls was obtained from Promega. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Maxi kit according to the manufacturer protocol (Qiagen, www.qiagen.com). The OD260/280 method on a photometer was employed to determine the appropriate DNA concentration. [94] Patient and control DNA samples were sonicated to produce a homogeneous smear DNA extending from approximately 600 bp to 2 Kb. DNA samples were then purified using the DNA Clean and Concentrator kit (Zymo Research, Orange, CA). Ten micrograms of genomic DNA both from the patient and from the control were sonicated. Test and reference DNA samples were subsequently purify using dedicated columns (DNA Clean and Concentrator, Zymo research, CA92867-4619, USA) and the appropriate DNA concentrations were determine by a DyNA Quant™ 200 Fluorometer (GE Healthcare).

3.2.2 Human oligonucleotides array

Array based CGH analysis was performed using commercially available oligonucleotide microarrays containing about 43,000 60-mer probes with an

[page 29:]


estimated average resolution of about 100-130 Kb (Human Genome CGH Microarray 44B Kit, Agilent Technologies) and microarrays containing 99,000 60-mer probes with an estimate average resolution of 50-65 Kb (Human Genome CGH Microarray 105A Kit, Agilent Technologies).


94. Sambrook, J. and M.J. Gething, Protein structure. Chaperones, paperones. Nature, 1989. 342(6247): p. 224-5.

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