This experimental approach combines BRDU incorporation with fluorescent in C two hybridization. To quantitatively analyze the replication timing of mammalian chromosomes. First, incorporate BRDU into live cells and harvest the metaphase chromosomes.
Then perform DNA fish combined with sequential BRDU antibody staining to identify the individual replicating chromosomes. Next, use photo microscopy to capture images of fish signals and BRDU incorporation in individual mitotic figures. Using cyto vision software, calculate the area and intensity of the pixels represented by the BRDU or DPI signals on each isolated chromosome.
Results can quantify and compare the BRDU incorporation in individual rearranged chromosomes and their holos. Because the method described here evaluates individual cells, it has the ability to detect changes in replication, timing on chromosomal rearrangements that are present in only a fraction of the cells in a population compared to the recently developed high throughput microarray and DNA sequencing protocols. This method allows for a direct comparison of homologous chromosomes residing within the same cell.
It also has the ability to identify unambiguously chromosome rearrangements that affect chromosome replication. Timing of entire chromosomes demonstrating the procedure will be Leslie Smith. A research associate in my laboratory Seed the cells at approximately 70%confluence in a 150 millimeter tissue culture dish and place in a cell incubator for 24 hours at the appropriate time.
Points prior to harvesting. Replenish the media with fresh complete media containing 10 micrograms per milliliter.BRDU. The length of time that cells are cultured in media with BRDU will vary with cell type and species.
Typically, G two phase is between two and five hours. Remove the culture media from the plate saving 10 milliliters in a 15 milliliter conical centrifuge tube. Rinse the cells with 10 milliliters of sine and remove the remaining sine by vacuum aspiration.
Then add five milliliters of 0.25%trypsin and incubate at room temperature until cells are detached from the plate. Harvest the cells and transfer the suspension to the 10 milliliters of reserved media. Pellet the cells by centrifugation, aspirate all but 0.5 milliliters of the media.
Now resus suspend the cells thoroughly using a past of pipette to osmotically swell the cells add three drops of 75 millimolar potassium chloride warmed to 37 degrees Celsius, and mix the cell suspension with a past of pipette. Then incorporate an additional 0.5 milliliters of hypotonic solution. Now bring the total volume of hypotonic solution to five milliliters and mix by pipetting.
Incubate the cell suspension at 37 degrees Celsius for 20 to 45 minutes depending on the cell type. Incubate the tubes at a slant to keep the cells in suspension. Then centrifuge at 400 times G for 10 minutes.
Aspirate all but 0.5 millimeters of the supernatant and use a past stir pipette to gently resus suspend the cell palette. The osmotically swollen cells will be fragile at this point, so care should be taken not to disrupt the cytoplasmic membranes. Next, add three drops of car noise, fixative and mix gently.
After incorporating an additional 0.5 milliliters of fixative, bring the volume of fixative to five milliliters and pipette to a homogenous cell suspension. These fixed cells can be stored in the dark at negative 20 degrees Celsius for several months. Harvest the fixed cells by centrifugation, aspirate the fixative and resuspend the pellet in 10 volumes of fixative holding a wet ice cold microscope.
Slide at approximately a 45 degree angle. Add the cell suspension dropwise to spread the cell suspension. Flood the slide with fixative and drain off the excess.
Then lay the slide flat on paper towels to air dry. Evaluate the samples for the presence of mitotic spreads using an inverted microscope. Add two microliters of 10 microgram per milliliter RNAs A in two XSSC to each slide and incubate at 37 degrees Celsius for one hour.
Perform three three minute washes of two XSSC pH 7.0 at room temperature. Then dehydrate the samples through an ethanol series at room temperature for three minutes. Each air dry the samples at room temperature to a chilled micro fuge tube.
Add four micrograms of DNA substrate labeled nucleotide, 10 X NIC translation buffer, DNTP mix and NIC translation enzyme. Incubate the reaction at 16 degrees Celsius overnight heat at 70 degrees Celsius for 10 minutes to stop the reaction. Then chill on ice for five minutes.
Now to ethanol precipitate the DNA add 48 microliters of three molar sodium acetate, 160 microliters of 0.25 micrograms per microliter caught 1D NA and 1.2 milliliters of 100%ethanol. Store the sample at negative 80 degrees Celsius for 10 minutes to overnight precipitate the DNA by centrifugation and wash the pellet with 70%ethanol resuspend the air dried DNA probe in 40 microliters of double distilled water for a final concentration of 100 nanograms per microliter of labeled back DNA for the back SEP simultaneous hybridization. Prepare two separate cot, 1D NA containing probe cocktails as detailed in the accompanying manuscript.
Denature the probe cocktails at 75 degrees Celsius for 10 minutes. Then incubate at 37 degrees Celsius for 30 minutes to a the cot 1D NA to repetitive sequences. Now combine the back probe with the S probe at a three to two ratio for 25 microliters per sample.
Slide next to denature the samples. Immerse the slides in 70%form amide two XSSC at 72 degrees Celsius and incubate for three minutes. Immediately dehydrate samples through an ethanol series at four degrees Celsius for three minutes.
Each air dry the samples at room temperature about 10 minutes before completion of the probe preparation. Equilibrate the slides on a 45 degrees Celsius slide warmer. Then add 25 microliters of the back S probe mix.
To each slide gently position a cover slip and seal along all edges with rubber cement for the hybridization step. Incubate samples overnight at 37 degrees Celsius in a humidified chamber. Perform three post hybridization washes of three minutes each in 50%IDE two XSSC pH 7.0 at 38 to 40 degrees Celsius.
The optimal temperature of the washes is probe dependent. If there's high background hybridization, then increase the temperature of the washes. Conversely, if the signal of the probe is weak and there's no background, then decrease the temperature Of the washes.
Perform one wash in PN buffer at room temperature for three minutes. For the BRDU detection, add 200 microliters of PNM blocking buffer to each slide. Incubate for 10 minutes at room temperature in the dark, drain the slides on a paper towel.
Then add 100 microliters of 50 micrograms per milliliter anti B-R-D-U-F-E conjugated antibody and incubate for 30 minutes At 37 degrees Celsius. Perform three three minute washes in PN buffer at room temperature. Remove the excess PN buffer off of each slide.
Then add 20 microliters of dappy Antifa mounting solution. Cover the slide with a paper towel and press down on the cover slip to force out air bubbles and excess mounting solution. Capture the images with a fluorescence microscope attached to a CCD camera.
Using the 100 x objective and automatic filter wheel and cyto vision software Capture BRDU using a fitzy filter and the chromosome paints back and centromere probes using a side three or Texas red filter. Also use a dappy filter for nuclei staining. Identify the individual chromosomes of interest with back EP or chromosome specific paint probes in the cyto vision software.
Cut out each chromosome of interest from the metaphase spread as a whole. Copy and paste the DPI and FITZY chromosome images into a new window. Select the graph icon in the tool menu, which allows the generation of a graph representing the area and intensity of both the Fitz and dappy staining along each individual chromosome.
This is done by drawing a line through the length of the chromosome from short arm to long arm. In the tool menu, select the data icon and generate a table displaying the calculated area and intensity for ZI and dpi. Next, select the ZI and DPI signals for the chromosomes of interest.
Then click the intensity and area buttons on the table icon. The software generates a numerical measurement of pixels per square inch for each signal. Next, the average pixel intensity of each chromosome is multiplied by the area occupied by those pixels.
To obtain the total number of pixels for the visualization of the latest replicating regions of chromosomes, evaluate the bandit pattern of BRDU incorporation for actively replicating regions of chromosomes. Then calculate the differences in replication timing between chromosome pairs based on differences in BRDU banding pattern. The chromosomes in mammalian cells replicate according to a temporal program with early and late replication occurring at the beginning and end of S-phase respectively.
A typical sase in mammalian cells lasts for eight to 10 hours, and G two is typically two to five hours. In this experimental system, BRDU is added for increasing periods of time to label the last portions of the chromosomes that replicate these cells contain a deletion of the human as a R six gene located at six Q 16.1. After a five hour treatment with BRDU mitotic cells were harvested and processed for BRDU incorporation and fluorescent in C two hybridization using a chromosome six paint probe, the two chromosome sixes display three fluorescent labels.
The significant difference in the BRDU banding pattern is consistent with the delay in replication timing of greater than two hours for one of the chromosome sixes. Cyto vision software depicts the difference signal intensity profiles for DPI and BRDU on the two chromosome sixes indicative of asynchronous replication. In addition, there is a significant difference in the total amount of BRDU incorporation when compared to a similar analysis of the DPI staining.
Inclusion of a back probe containing the human as AR six gene highlights the single chromosome six containing a deletion on its long arm in this cell. Interestingly, there is more intense BRDU incorporation and a more extended banding pattern of BRDU incorporation than on the non-ED chromosome six. When mitotic spreads from seven different cells were processed for quantification of the replication timing difference between chromosome sixes.
The BRDU levels show a greater than twofold difference between the total pixel values for the deleted and non-ED chromosome sixes. Once mastered, this technique can be performed properly in three days. We hope you now have a good understanding of how to measure replication, timing of homologous chromosomes residing within the same cell, and to unambiguously identify chromosomal rearrangements that correlate with alterations in replication.
Timing of entire chromosomes.
