Technische demonstratie van Whole Genome Array comparative genomic hybridisatie

The following short presentation is a video showing the hybridization protocol for the 27 K sub megabase ting array. This array or smart array is composed of 26, 946 individually sequenced back clones printed on a single slide. They are spotted in duplicate for a total of 54, 000 elements per slide printed in an 18 by 54 millimeter area.

The resulting data is comparable to high density oligo arrays without requiring data interpretation by bioinformatics. One major advantage of using a large insert array such as back clones is that it allows the use of as little as 100 nanograms of sample DNA without amplification. This allows the analysis of a variety of samples such as DNA from blood frozen tissue sorted cells, and FFPE material.

The smart array has been proven useful in the clinical setting. At the BC Cancer Agency, multiple patient sample analysis is easily accommodated into the average work week. For cytogenetics.

Most of the steps used in array CGH are similar to conventional CGH or fish. Allowing quick introduction of the protocol into Established labs. The quality of DNA is the single greatest influence on results in array's.

CGH hybridization, poor quality or contaminated DNA will adversely affect PSI incorporation into the product resulting in dim images or noise in the data. The NanoDrop spectrophotometer is an excellent tool to assess the DNA quality prior to hybridization. To quantitate the DNA set, the NanoDrop to measure nucleic acid and the mode to measure DNA 50 blank, the NanoDrop with 1.5 microliters of the same solution as your resuspended sample.

In this case, it is water Tris. EDTA has been shown to have potential negative effects on PS D incorporation. Remove the blank wipe with a Kim wipe and add 1.5 Microliters of your sample measure and Record your sample concentration.

The two 60 over two 80 reading on the NanoDrop is a good indicator of DNA quality where a 1.8 ratio is optimum. The double ratio method two 60 over two 80 and two 60 over two 30 provides an even better way to determine the purity of your samples. The NanoDrop displays a curve representing the absorbance relative to the wavelength.

A smooth curve with no peaks is indicative of good quality DNA wipe and clean the NanoDrop with water and a Kim wipe, or if the sample is limiting, pipette the Sample off the pedestal. This Protocol is optimized for between 100 to 400 nanograms of sample. 200 nanograms is the target amount for good quality DNA To label the sample the following reagents and equipment are required.

Buffer SCI three SCI five random ERs 10 times DNTP mix reference DNA sterile water 0.2 microliters sterile PCR tubes ice heat block at 100 degrees Celsius incubator at 45 degrees Celsius. Set up one reaction tube for reference DNA and one reaction tube. For sample DNA, they will be labeled with sci-fi and SCI three respectively.

In a research setting, we prefer to use CY three for our sample DNA as it is more resistant to ozone Degradation. Combine Your DNA water and canal buffer to the sample and repeat for the reference DNA. In our lab, we combine the 10 times buffer with the random Optimus.

To create a five time solution, add sterile water to a total volume of 17 microliters. Transfer the tubes to a heat block, denature for five minutes at 100 degrees Celsius. Transfer immediately To ice.

Add Four microliters of 10 times DNTP mix. Add the side eyes. Add two microliters of SCI three labeled DCTP to sample DNA.

Add two microliters of scifi labeled DCTP to reference DNA. Add 2.5 microliters of canal and mix. The solution can either be hand mixed or vortex.

Briefly spin the solution down to the bottom of the tube with a quick pulse in a benchtop centrifuge. Transfer to an oven and incubate at 37 degrees overnight. The overnight hybridization time can be adjusted between 14 to 24 hours without adversely affecting the labeling process.

To fit a laboratory Schedule using a MicroCon YM 30 column add 100 microliters caught 1D NA to the column. Be careful not to touch the membrane with a pipette tip caught. DNA batch to batch variance is known to occur even when purchased from major distributors.

It is recommended that large batches are purchased and tested prior to use. There will now be two tubes for each sample hybridization, one colored blue and one colored red. Combine the tubes for each sample and mix by pipetting.

Then transfer the entire solution to the MicroCon membrane. Place the column in the Provided MicroCon tube and spin at 13, 000 G for 10 minutes. The MicroCon column is a size exclusion column and will trap the DNA with the incorporated side dyes and allow the unincorporated dyes to wash through the membrane.

To Wash any residual unincorporated nucleotides or side, add 200 microliters sterile distilled water to the Membrane. Spin again at 13, 000 G For 10 minutes. Discard the tube and add 45 microliters of hybridization solution to the top of the MicroCon concentrator column.

The hybridization solution that we use is Dig Easy from Roche Scientific five microliters shared herring sperm may also be added to the solution. Traditionally, this was used to block non-competitive binding to the slide surface. The advent of new binding substances has allowed the elimination of this step.

However, it is sometimes added to increase the viscosity of the hybridization solution. Invert the MicroCon column and place in a new labeled tube. Spin the tube at 3000 G for three minutes.

The solution will pool on the bottom of the new tube. 1.5 microliters of the solution will be used for quantification of the SD Incorporation. Set the NanoDrop To microarray analysis, blank.

The NanoDrop with 1.5 microliters of dig. Easy hybridization Buffer. Measure the blank with the NanoDrop.

The reading should be zero. Remove the blank wipe with a kim wipe and add 1.5 microliters of your sample measure And record your sample incorporation. The NanoDrop will give you a PICA mole per microliter concentration of both the S3 and sci-fi dye incorporations samples with incorporations below.

Three ole per microliter should be considered to have too few Fluor fours To continue, the NanoDrop will display a graph of the absorbance versus the wavelength. Two peaks should be visible, one for each of the side eyes wipe and clean the NanoDrop With water and a kim wipe. Place the cover Slip on a slide warmer or heat block prewarm to 45 degrees Celsius to maintain hybridization Temperature.

Place 42 microliters of probe solution onto the 22 millimeter by 60 millimeter cover slip. Make sure that there are no air bubbles in the solution. A quick trick to remove a stubborn bubble is to take a fresh cover slip and pop the bubble with a sharp corner.

Align the slide over the cover slip and probe solution. Lower one edge of the slide, allowing contact with the hybridization solution. Continue to lower one edge until the cover slip attaches to the slide with surface tension.

Invert the slide. Place the slide into a hybridization cassette, prewarm to 45 degrees Celsius, and add 10 microliters of water in the lower groove to control humidity. This step should be practiced as the dig easy crystallizes at lower temperatures and will leave background on the slide.

This is especially important when the solution is on the slide as it is a very thin layer of liquid. At this point, seal the cassette and incubate for 36 to 40 hours At 45 degrees Celsius. Removal Of the slide from the hybridization cassette is critical.

Dig, easy crystallizes at room temperature. The slide should be immediately immersed and the cover slip removed in the wash solution. All wash solutions should be pre-made and have a pH of seven non neutral pH may degrade the side eyes.

Prewarm the wash solution to 45 degrees Celsius. Add approximately 60 milliliters of the wash solution to a Copeland jar prior to opening the hybridization chamber. Open the chamber.

Remove the slide with tweezers and add the slide to the wash solution. With the cover slip still attached, wait 10 to 20 seconds and retrieve the cover slip with tweezers. It should have fallen off the slide.

This is an important step as it prohibits the dig easy buffer from crystallizing as well as reduces any potential scratching of the slide by mechanical removal of the cover slip. Wash the slide three times in wash solution. One minute each with agitation.

Rinse the slide three times. There should be no residual Bubbles from the SDS in the wash solution visible after the last wash. Leave the slide in the wash solution until ready to Centrifuge.

Centrifuge In a 50 milliliter Falcon tube at 700 G for one minute. Make sure the Falcon tube is dry prior to use. Immediately scan the slide as signal intensities will diminish over time.

Depending on the ambient ozone Levels, every Scanner is different, but there are some common guidelines to follow. For slide imaging. The ambient ozone levels are critical during the scanning process.

Sci-fi die is ozone sensitive and will degrade rapidly during a long scan time. It has been shown that five parts per billion of ozone affects S five. This is equivalent to light traffic pollution on a clear day.

Ozone meters are recommended to record ambient ozone levels. A separate image is created for each channel S3 and SCI five. The channels can be balanced by altering the exposure, time, excitation, input, or capture sensitivity of the scanner.

These images are now ready for analysis.