Editing Comparative genomic hybridization (section)

==Basic methods==

===Metaphase slide preparation===
The DNA on the slide is a reference sample, and is thus obtained from a karyotypically normal man or woman, though it is preferential to use female DNA as they possess two X chromosomes which contain far more genetic information than the male Y chromosome. Phytohaemagglutinin stimulated peripheral blood lymphocytes are used. 1mL of heparinised blood is added to 10ml of culture medium and incubated for 72 hours at 37&nbsp;°C in an atmosphere of 5% CO<sub>2</sub>. Colchicine is added to arrest the cells in mitosis, the cells are then harvested and treated with hypotonic [[potassium chloride]] and fixed in 3:1 [[methanol]]/[[acetic acid]].<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

One drop of the cell suspension should then be dropped onto an ethanol cleaned slide from a distance of about 30&nbsp;cm, optimally this should be carried out at room temperature at humidity levels of 60–70%. Slides should be evaluated by visualisation using a phase contrast microscope, minimal cytoplasm should be observed and chromosomes should not be overlapping and be 400–550 bands long with no separated [[chromatids]] and finally should appear dark rather than shiny. Slides then need to be air dried overnight at room temperature, and any further storage should be in groups of four at −20&nbsp;°C with either [[Silicon dioxide|silica]] beads or [[nitrogen]] present to maintain dryness. Different donors should be tested as hybridization may be variable. Commercially available slides may be used, but should always be tested first.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

===Isolation of DNA from test tissue and reference tissue===

Standard [[phenol extraction]] is used to obtain DNA from test or reference (karyotypically normal individual) tissue, which involves the combination of [[Tris]]-[[Ethylenediaminetetraacetic acid]] and [[phenol]] with [[aqueous solution|aqueous]]  DNA in equal amounts. This is followed by separation by agitation and centrifugation, after which the [[aqueous solution|aqueous]] layer is removed and further treated using [[diethyl ether|ether]] and finally [[ethanol precipitation]] is used to concentrate the DNA.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

May be completed using DNA isolation kits available commercially which are based on [[Affinity chromatography|affinity columns]].<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

Preferentially, DNA should be extracted from fresh or frozen tissue as this will be of the highest quality, though it is now possible to use archival material which is formalin fixed or paraffin wax embedded, provided the appropriate procedures are followed. 0.5-1&nbsp;μg of DNA is sufficient for the CGH experiment, though if the desired amount is not obtained DOP-PCR may be applied to amplify the DNA, however it in this case it is important to apply DOP-PCR to both the test and reference DNA samples to improve reliability.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

===DNA labelling===

[[Nick translation]] is used to label the DNA and involves cutting DNA and substituting nucleotides labelled with fluorophores (direct labelling) or biotin or oxigenin to have fluophore conjugated [[Antibody|antibodies]] added later (indirect labelling). It is then important to check fragment lengths of both test and reference DNA by [[gel electrophoresis]], as they should be within the range of 500kb-1500kb for optimum hybridization.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

===Blocking===
Unlabelled Life Technologies Corporation's Cot-1 DNA (placental DNA enriched with repetitive sequences of length 50bp-100bp)is added to block normal repetitive DNA sequences, particularly at [[centromeres]] and [[telomeres]], as these sequences, if detected, may reduce the fluorescence ratio and cause gains or losses to escape detection.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

===Hybridization===
8–12μl of each of labelled test and labelled reference DNA are mixed and 40&nbsp;μg Cot-1 DNA is added, then precipitated and subsequently dissolved in 6μl of hybridization mix, which contains 50% formamide to decrease DNA melting temperature and 10% dextran sulphate to increase the effective probe concentration in a saline sodium citrate (SSC) solution at a pH of 7.0.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

[[Denaturation (biochemistry)|Denaturation]] of the slide and probes are carried out separately.  The slide is submerged in 70% formamide/2xSSC for 5–10 minutes at 72&nbsp;°C, while the probes are denatured by immersion in a water bath of 80&nbsp;°C for 10 minutes and are immediately added to the metaphase slide preparation. This reaction is then covered with a coverslip and left for two to four days in a humid chamber at 40&nbsp;°C.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

The coverslip is then removed and 5 minute washes are applied, three using 2xSSC at room temperature, one at 45&nbsp;°C with 0.1xSSC and one using TNT at room temperature. The reaction is then preincubated for 10 minutes then followed by a 60-minute, 37&nbsp;°C incubation, three more 5 minute washes with TNT then one with 2xSSC at room temperature.  The slide is then dried using an ethanol series of 70%/96%/100% before counterstaining with DAPI (0.35 μg/ml), for chromosome identification, and sealing with a coverslip.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

===Fluorescence visualisation and imaging===
A [[fluorescence microscope]] with the appropriate filters for the [[DAPI]] stain as well as the two fluorophores utilised is required for visualisation, and these filters should also minimise the crosstalk between the fluorophores, such as narrow band pass filters. The microscope must provide uniform illumination without [[Diatonic and chromatic|chromatic]] variation, be appropriately aligned and have a "plan" type of objective which is [[apochromat]]ic and give a magnification of x63 or x100.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

The image should be recorded using a camera with spatial resolution at least 0.1&nbsp;μm at the specimen level and give an image of at least 600x600 pixels. The camera must also be able to integrate the image for at least 5 to 10 seconds, with a minimum photometric resolution of 8 bit.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

Dedicated CGH software is commercially available for the image processing step, and is required to subtract background noise, remove and segment materials not of chromosomal origin, [[Normalization (statistics)|normalize]]  the fluorescence ratio, carry out interactive karyotyping and chromosome scaling to standard length. A "relative copy number karyotype" which presents chromosomal areas of deletions or amplifications is generated by averaging the ratios of a number of high quality metaphases and plotting them along an ideogram, a diagram identifying chromosomes based on banding patterns. Interpretation of the ratio profiles is conducted either using fixed or statistical thresholds ([[confidence intervals]]). When using confidence intervals, gains or losses are identified when 95% of the fluorescence ratio does not contain 1.0.<ref name="Weiss,Hermsen,Meijer,VanGrieken,Baak,Kuipers,VanDiest" />

=== Extra notes ===
Extreme care must be taken to avoid contamination of any step involving DNA, especially with the test DNA as contamination of the sample with normal DNA will skew results closer to 1.0, thus abnormalities may go undetected. FISH, [[Polymerase chain reaction|PCR]]  and [[flow cytometry]] experiments may be employed to confirm results.<ref name="Pinkel,Albertson" /><ref name="Evangelidou,Alexandrou,Moutafi,Ioannides,Antonios,Koumbaris,Kallikas,Velissariou,Sismani,Patsalis">Evangelidou P, Alexandrou A, Moutafi M, Ioannides M, Antoniou P, Koumbaris G, Kallikas I, Velissariou V, Sismani C, Patsalis PC (2013) Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature. BioMed Research International 2013.</ref>