Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

The overall goal of this procedure is to determine the length of telomeres in eukaryotic cells using a modified version of a telomere restriction fragment analysis procedure, that's described by Doctors Jerry W.Shay, Woodring Wright, and colleagues. By providing data on the telomere length, the procedure can answer questions on cell biology, behavioral medicine, epidemiology, and infectious diseases, on the effects of age, stress, drug treatments, and infections. The main advantage of this technique is that it provides a direct measurement in kilobases of the average telomere length in a variety of cells.

The genomic DNA samples to be analyzed were extracted from cells using a commercial DNA extraction kit as described in the text protocol. Perform the digestion of the genomic DNA in a final volume of 20 to 40 microliters. Combine the following in each microcentrifuge tube:three micrograms of genomic DNA;10x DNA digest buffer;one microliter of Rsa1 restriction enzyme;one microliter of Hinf1 restriction enzyme;and sterile, deionized water to the final volume.

Centrifuge briefly to ensure that all components are in the bottom of the tube. Incubate the digestions at 37 degrees Celsius, for at least 16 hours. Begin this procedure by preparing 0.6%agarose solution as described in the text protocol.

Prepare the gel electrophoresis system for gel casting. Attach the end gates to the gel casting tray. Once the agarose gel solution has cooled to 50 degrees Celsius, pour it into the gel casting tray, place a cone into the gel, and allow the gel to harden for 45 minutes uncovered at room temperature.

Remove the end gates from the casting tray, and remove the comb from the gel. Fill the gel electrophoresis system, with electrophoresis buffer to the fill line, ensuring that the gel is completely submerged in the buffer. Load the wells of the gel with the DNA samples, leaving a blank well between samples.

Add 10 microliters of a DNA ladder into the wells on the opposite ends of the gel. Run the gel electrophoresis at 150 volts for 30 minutes, to quickly move the DNA into the gel. After 30 minutes, adjust the voltage to 57 volts, and run for 18 to 24 hours.

After the gel has run for 18 to 24 hours, turn off the elecctrophoresis source and remove the gel casting tray with the gel from the elecctrophoresis buffer. Slice of the top right corner of the gel to serve as a reference for the orientation of the gel. Cut a piece of filter paper to a size slightly larger than the gel.

Place the filter paper on top of the gel in a casting tray, and allow the paper to soak up the excess solution on the gel. Carefully remove the air bubbles between the filter paper and the gel, using a pipet as a roller to squeeze the bubbles out through the sides. Remove the gel from the casting tray by flipping the gel and filter paper over so that the paper is underneath the gel.

Transfer the gel with the filter paper to a gel dryer, and cover the gel with plastic wrap. Remove all air bubbles between the plastic wrap and gel using a pipet as a roller. Dry the gel at 50 degrees Celsius for two hours.

Once the gel has dried, remove the plastic wrap and transfer the gel and filter paper to a glass dish containing 250 milliliters of deionized water. Carefully remove the filter paper, and incubate the gel at room temperature for 15 minutes. Lay the gel on top of a 12 by 12 inch nylon mesh.

Roll the nylon mesh and gel together making sure that the gel is not in contact with itself. Place the mesh and gel into a hybridization tube. Combine 100 milliliters of 5x SSC and 12 microliters of green fluorescent nucleic acid stain, and place into the hybridization tube.

Incubate for 30 minutes at 42 degrees Celsius in a hybridization oven with rotation. Protect the solution from light. After 30 minutes, remove the nylon mesh and gel from the hybridization tube.

Roll open and place flat into a glass dish containing 250 milliliters of deionized water. Gently remove the nylon mesh. Image the gel using a phosphorimager with the indicated settings.

Prepare the radioactive telomere probe following the facility's regulations for handling radioactivity. In the microcentrifuge tube combine the following:two microliters of telomere probe;2.5 microliters of 10x polynucleotide kinase reaction buffer;three microliters to ATP labeled on the gamma phosphate group with P32;four microliters of T4 polynucleotide kinase;and DNase free, sterile, deionized water to a final volume of 20 microliters. Briefly centrifuge the tube.

Incubate the tube in a water bath at 37 degrees Celsius for one hour. Centrifuge the tube briefly, and incubate at 65 degrees Celsius for 20 minutes to stop the reaction. Load the radioactive probe solution into a prepared G25 chromatography column, and centrifuge for two minutes at 700 times g.

Save the filtrate, which contains the radioactive telomere probe. Place the gel into a glass dish containing 250 milliliters of Denaturing Solution and incubate for 15 minutes at room temperature. Remove the Denaturing Solution and replace with 250 milliliters of sterile, deionized water.

Incubate for 10 minutes on an orbital shaker at room temperature. Remove the deionized water. Replace with 250 milliliters of neutralization solution and incubate for 15 minutes at room temperature.

Roll the gel into a nylon mesh, and place into the hybridization tube. Add 20 milliliters of the hybridization solution, and incubate in a hybridization oven at 42 degrees Celsius for 10 minutes with rotation. After 10 minutes, remove the hybridization solution and replace with 20 milliliters of fresh hybridization solution.

Add the entirety of the telomere probe, and incubate in a hybridization oven at 42 degrees Celsius with rotation overnight. When the hybridization with the telomere probe is complete remove the gel and mesh from the hybridization tube, and place into a glass dish, containing 250 milliliters of 2x SSC. Unroll and remove the nylon mesh from the dish.

Place the glass dish onto an orbital shaker and incubate for 15 minutes at room temperature. Remove the 2x SSC, replace with 250 milliliters of 0.1x SSC, plus 0.1%SDS solution, and incubate for 15 minutes on the orbital shaker at room temperature. Remove the 0.1x SSC plus 0.1%SDS solution.

Replace with 250 milliliters of 2x SSC and incubate for another 15 minutes on the orbital shaker. Remove the gel from the 2x SSC, and wrap the gel in plastic wrap. Remove all bubbles and air pockets between the gel and the plastic wrap.

Place the gel into an exposure cassette with a phosphor screen, and expose the phosphor screen to the gel overnight. On the following day, use a phosphor imager to image the exposed phosphor screen. The phosphor images are subsequently used for telomere length quantification as described in the text protocol.

Three concentrations of DNA isolated from an immortalized human foreskin fibroblast cell line and human peripheral blood mononuclear cells were analyzed for telomere length. This phosphor image of the dried agerose gel stained with green fluorescent nucleic acid stain shows the location of the DNA ladder bands in lanes one and eight. The distance measured from the top of the gel to each molecular weight standard is indicated by blue arrows.

Following hybridization with a telomere probe, the telomere bands appear as smears in each lane. Grids of 150 boxes, were calculated for each lane plus one background lane. The selected areas of analysis for each set of samples are shown.

Separate background lanes marked B were selected for each set of samples, to ensure the background intensities were similar for each sample set. Average telomere length calculations revealed a higher average telomere length in the immortalized cell line than in human peripheral blood mononuclear cells. These results also demonstrate that the amount of genomic DNA analyzed in each lane is critical to obtaining a detectable signal following hybridization.

Once mastered, this technique can be performed in 12 hours spread out over 2 1/2 days, with two overnight incubations if it's performed properly. While attempting this procedure, it's important to remember to use at least three micrograms of DNA for each sample, and to measure each sample in duplicate. After its development, this technique paved the way for researchers in the field of aging to explore mechanisms responsible for maintaining telomere length in different animal species, including humans.

After watching this video, you should have a good understanding of how to directly determine the average length of telomeres themselves. Don't forget that working with radioactivity can be extremely hazardous, and precautions such as wearing lab coats and gloves should always be taken while performing this procedure.