Array comparative genomic hybridisatie (Array CGH) voor detectie van genomische kopij Varianten

The overall goal of this procedure is to array comparative genomic hybridization to identify imbalances in the genome that can lead to a wide variety of genetic diseases. This is accomplished by first labeling the patient's DNA with a fluorescent dye. In the second step, the patient, DNA and a differently labeled reference DNA, are hybridized to the array.

Next, the array is scanned to measure the amount of the patient and reference DN abound to each probe. In the final step, the scanned image is processed to identify the regions of the genome where the patient's DNA has more or less material than the reference DNA ultimately array. Comparative genomic hybridization is used to determine whether the patient carries a copy number variant that results in a genetic syndrome.

Visual demonstration of this method is critical as assembly of the array slide can be difficult and it is easy for the hybridization. Mix the spill out of the chamber Before beginning the labeling reaction. Thaw the ready-to-use 96 well plate of nucleotides and primers at four degrees Celsius and protected from light for about an hour.

Once thawed, equilibrate the covered plate for another 30 minutes at room temperature, equilibrate the DNA samples for 15 minutes at 60 degrees Celsius at this time as well. While the samples are warming, use a liquid handling robot to dispense enough nuclease free water into each well of a new 96 well plate. Then when the DNA is ready transfer one microgram of sample per well to the 96 well plate of water.

Now transfer 20 microliters of the equilibrating nucleotides and primers to each one of the plate containing the diluted DNA samples and seal the plate with strip caps taking care to make a tight seal. Next, denature the DNA at 99 degrees Celsius in a PCR machine with a heated lid after 10 minutes and knee the primers by snap cooling the plate on ice for five minutes. Then use the liquid handling robot to add 10 microliters of clean out exo DNA polymerase enzyme to each sample.

Mix the samples by pipette and then seal and incubate the plate at 37 degrees Celsius for 16 hours. The next day terminate the reaction with five microliters of stop buffer per well Then remove the unincorporated nucleotides. Transfer the contents of each well to individual pre-labeled two milliliter tubes.

Load the samples and DNA purification spin columns into a spin column processing robot, as well as the appropriate associated buffers. According to the manufacturer's instructions, the spin column processing robot will bind the labeled DNA to the silica membrane with 250 microliters of high salt DNA binding buffer per tube, after which the impurities will be removed from the membranes with two washes of 500 microliters of wash buffer each. The robot will then add 15 microliters of low salt solution buffer to the membranes to recover the purified labeled DNA samples in approximate 12 microliter volumes to hybridize the samples.

Next preheat a hybridization oven to 65 degrees Celsius and prewarm the backing slides and hybridization chambers. To prepare the hybridization mix, combine 1.1 microliters of cot 1D NA 4.95 microliters of the manufacturer supplied blocking mix, and 24.75 microliters of hybridization buffer. Use the liquid handling robot to allocate this mix into each well of a new 96 well plate pre wetting each tip to enhance the transfer accuracy.

Next pipette 9.35 microliters of the appropriate signing, three labeled DNA, followed by 9.35 microliters of the appropriate signing, five labeled DNA. To each well seal the plate, fourex the samples for one minute, and give the plate a quick spin to collect the contents at the bottom of each. Well denature the labeled DNA in a pre hybridization incubator.

First for three minutes at 95 degrees Celsius, followed by 30 minutes at 37 degrees Celsius. Then working on a 42 degrees Celsius heated platform. Place the backing slide into the hybridization chamber, ensuring that the transparent part of the gasket aligns with the windowed part of the hybridization chamber Using pre-wet tips.

Now slowly pipette 42 microliters of the hybridization mix into the center of the appropriate position of the array backing slide. Taking care that the liquid does not touch the rubber ring boundary. Once all the positions are filled carefully lower the array slide onto the backing slide and assemble the hybridization chamber.

Taking care that the side of the array with writing is facing the backing slide, then tighten the hybridization chamber screw fully and inspect the assembled hybridization chamber, confirming that there is no leakage of hybridization. Mix outside of the rubber ring boundaries and that each air bubble is approximately four millimeters in height. When the hybridization chamber is rested on its end vertically, rotate the hybridization chamber to check this.

All the air bubbles in each position are moving. If any of the bubbles are stuck, give the chamber a sharp tap on the bench. Then place the hybridization chambers into the rotating oven at 65 degrees Celsius for 24 hours.

The next day submerge the hybridization chambers in wash buffer one, and use a pair of plastic flat edged forceps to pry the slides apart. Then discard the gasket slides and place the array slides into a rack submerge in fresh buffer one. Wash the array slides in approximately 700 milliliters of wash buffer for one to five minutes, stirring vigorously with a flea and a magnetic starr.

Then transfer the array slides to around 700 milliliters of wash buffer, two for 90 seconds with more vigorous stirring at the end of the second wash. Gently lift the array slides outta the buffer. They should emerge dry.

Then load the array slides into the slide holders at the scanner along with the slide protectors, and then scan the samples. According to the array manufacturer's instructions, each probe on a hybridized array is visualized as a mixture of red and green fluorochromes. The ratios of red to green fluorescent signal for each probe are quantified by the scanner and the associated software lots them as log two ratios according to their genomic position.

The resulting array traces allow interpretation of regions identified as genomically unbalanced. For example, in this trace from a child with Williams syndrome, a recurring micro deletion syndrome mediated by low copy repeats in the proximal region of chromosome seven, the genomic imbalance was identified by the software with a red line. The prop fluorescent log ratio should cluster closely around zero, indicating a green to red ratio of one to one for normal regions of the genome.

Scattered array traces as observed in this scan, may result in inaccurate calling of the abnormal regions, or a failure to identify the genomic imbalance and may be caused by a number of factors, including poor DNA quality, or the presence of rays, levels of ozone in the atmosphere sphere. After watching this video, you should have a good understanding of how to process patient samples for testing by A CGH and to produce good quality data for downstream analysis and detection of copy number variation.