The overall goal of the following experiment is to examine the telomere G overhang structure. In trap brucey cells. Native in gel hybridization uses a sequence specific oligo probe to hybridize to the single stranded region of the telomere under the native condition.
This single stranded region is the telomere G overhang region and therefore the amount of hybridized oligo probe represents its length adapter. Ligation allows the ending sequence of the telomere G overhang to be determined when the unique oligo is labeled. This method involves the ligation of a sequence specific adapter to the end of the telomere G overhang structure for the ligation mediated primary extension assay.
The guide oligo is end labeled and anal to the unique oligo before it is ligated to the chromosome ends. After ligation, primary extension is performed using T four DNA polymerase. The length of the G overhang can then be determined as the product is n minus six nucleotides longer than the actual G overhang where N represents the length of the guide oligo in the adapter.
Hi, I am bi Bo, an assistant professor in the Department of Biological, geological and Environmental Sciences at Cleveland State University. Hey, I'm Gio. I am graduate student in Dr.Lee's lab, and today we are going to show you a procedure to analyze geo hank structure.
We use this procedure to study telomere structure and functions in Trapasso while working with radioactivity. As in this experiment, it is necessary to take proper safety precautions. Institutional environmental health and safety department should be consulted before this is attempted.
In this video, some of these precursors are absent for demonstration purpose only. So let's get started. The principle of this part of the procedure is that under native conditions, an unlabeled tell C probe comprised four repeats of C-C-C-T-A.
A oligo can only hybridize to the single stranded telomere G overhang region. The hybridization intensity is thus proportional to the length of the overhang. After denaturation and neutralization, the same probe will be able to hybridize to the whole telomere region.
The intensity of this hybridization represents the total amount of telomere DNA and is used as a loading control. The final G overhang level can then be calculated by dividing the native hybridization signal by that obtained after denaturation. This procedure begins with a separation of intact chromosomes by past field gel electrophoresis or PFGE, which requires that genomic DNA Be prepared in agros plugs to avoid mechanically shearing the DNA to accomplish this first dissolve low melting point, or LMP agros in L buffer to 1.6%Keeping it warm at 50 degrees Celsius, then harvest 200 million T brucey cells by centrifugation.
Remove the supinate and resuspend the cell pellet with L buffer to a final concentration of 5 million cells per milliliter. Incubate the cell suspension between 42 and 50 degrees Celsius for 10 minutes. Then add one volume of 1.6%LMP to one volume of cells and quickly mix well aliquot 85 microliters of the cell suspension to each well of a disposable plug mold.
Once the plugs are solidified, transfer them into a 15 milliliter conical tube with five milliliters of L buffer and 1%sarco cell. Add a pinch of proteinase K powder. Mix well and incubate at 50 degrees Celsius for 48 hours.
Following incubation, discard the buffer and wash the plugs with five milliliters of L buffer twice. Then repeat the proteinase K digestion in L buffer stroke SAR caol for another 48 hours. Wash the plugs with fresh L buffer again and store them at four degrees Celsius in L buffer.
These plugs should be used within two weeks. Prepare the agros gel with the DNA plugs as described in the written protocol. Next, run the gel in a chef DR two unit.
Following PFGE, the gel is stained des stain photographed and dried. Once the gel is dry, place the gel in a hybridization bag and add 20 milliliters of pre hybridization buffer. Remove as much air from the bag as possible and seal the bag.
Incubate the hybridization bag in a water bath at 50 degrees Celsius with a gentle rocking for at least one hour while pre hybridizing the gel label. The oligo probes incubate the reaction at 37 degrees Celsius for one hour. Then add 90 microliters of TNES buffer to the reaction mixture.
To purify the labeled probe, load the probe on top of a pre-made G 25 column. Wash it with 700 microliters of TNAS buffer and elute the probe in 600 microliters. Following gel pre hybridization, boil the labeled probe for five minutes and add it to 25 milliliters of fresh pre hybridization buffer.
To make the hybridization buffer, remove the pre hybridization buffer from the bag filter. Sterilize the hybridization mixture with a 0.22 micrometer syringe filter and add it directly into the hybridization bag. Seal the bag and put it in a shallow container with warm water.
Place the whole container into the 50 degrees Celsius water bath and incubate it overnight with gentle rocking. The next day, cut a small opening in the hybridization bag into a 50 milliliter conical tube. Pour out as much of the hybridization mixture as possible.
Keep the probe frozen at minus 20 degrees Celsius. Remove the gel from the hybridization bag and put it into a container wash and dry the gel as described in the written protocol. Once dry, wrap the gel with plastic wrap and expose it to a phospho imager for approximately three days.
After three days, scan the phospho imager to perform the hybridization after denaturation. Begin by incubating the gel in denaturing buffer at room temperature for 30 minutes. Then wash the gel and neutralization buffer for 30 minutes.
Next, rinse the gel in distilled water for three minutes. Pre hybridize the gel at 55 degrees Celsius for one hour. Repeat the hybridization using the same probe containing hybridization mixture as before, but at 55 degrees Celsius overnight following hybridization.
Wash and dry the gel as before. Then wrap the gel and expose it to a phospho imager for approximately two hours. Scan the phospho imager Finally normalize the hybridization signal obtained before denaturation with that obtained after denaturation.
In this part of the procedure, the T the mere terminal nucleotide is determined by adapter ligation. The unique oligo is end labeled anil to the guide oligos and ligated to the telomere end. Only when the unique guide adapter bears a three prime overhang that is compatible with the terminal telomere sequence will the adapter be ligated.
For T brucey. Telomeres may terminate in one of six different nucleotides of the TT, A GGG repeats. Hence six different guide oligos are used to begin this process.
Treat the unique oligo with kinase by mixing six microliters of 10 mol per microliter of unique oligo with three microliters of 10 times PNK buffer, five microliters of gamma P 32 A TP 14 microliters of double distilled water and two microliters of T four PNK. Incubate the mixture at 37 degrees Celsius for one hour. Next, use the kayak quick nucleotide removal kit to remove the unincorporated hot A TP and elute.
The end labeled oligo with 60 microliters of elution buffer. To obtain a final concentration of about one P mol per microliter to create the adapter a kneel the unique oligo to the guide oligo. Add 10 microliters of purified unique oligo to two microliters of each guide oligo and one microliter of a kneeling buffer.
Put the tubes into an 85 degrees Celsius heat block and turn off the heat block. Let the tubes and heat block cool to RI temperature for a few hours. Then ligate the adapters to the total genomic DNA.
Incubate the mixture at 16 degrees Celsius in a PCR machine Overnight. Following an overnight incubation, perform a restriction endonuclease digestion as described in the written protocol. Add three microliters of 10 times orange G dye to each sample.
Load the DNA samples on a 20 by 20 centimeter 0.7%agros gel. Run the gel at 30 volts for 30 minutes. Then continue with higher voltage, keeping the voltage less than 120 volts for a total of 1000 volts in one hour.
After the gel has run, scan the AUM bromide stain gel with the typhoon scanner. Finally dry the agros gel and expose the dried gel to the phospho imager overnight. In ligation mediated primer extension, the guide oligo is end labeled and anil to the unique oligo before it is ligated to the chromosome ends only.
The unique guide adapter bearing a three prime overhang compatible with the G overhang will be ligated. After removal of the unligated oligo pairs primary extension can be carried out with T four DNA polymerase, which lacks strand displacement and five prime to three prime endonuclease activity. The final length of the extended guide oligo therefore reflects the length of the G overhang.
To begin the ligation mediated primer extension kinase, treat the unique oligo and the guide oligo incubate both mixtures at 37 degrees Celsius for 60 minutes. Aneel the guide oligo and the unique oligo by combining 10 microliters of the phosphorylated, unique oligo and 10 microliters of the end labeled guide oligo in a 1.5 milliliter einor tube, Anil the oligos and ligate the Anil guide. Unique oligo adapter to the telomere ends as described earlier, precipitate the DNA and perform primary extension as described in the written protocol.
Prepare a 10%acrylamide, seven molar urea one times TPE gel for running the extension products. Load four microliters of each pre boiled sample with added dye onto the gel. Run the gel at 800 volts in one times TBE until the blue dye reaches the bottom of the gel.
Subsequently take the gel off the glass plate. Set the gel dryer to keep at 65 degrees Celsius for 30 minutes and dry the gel keep drying for 30 more minutes after the gel dryer turns back to room temperature finally exposed to the phospho imager for two hours. Intact tbrc chromosomes separated by PFGE are shown.
T brucey cells normally contain approximately 100 copies of mini chromosomes, all with similar size that migrate to the same position on the gel. Due to this fact after hybridization with TC oligo probe, the telomere G overhang signal is most prominent on mini chromosomes. After denaturation and neutralization hybridization with TLC oligo probe reveals telomere signals on all chromosome ends.
Hybridization with TLG oligo probe normally does not yield any hybridization signal because T brucey cells do not have telomere C overhang structures. After denaturation and neutralization hybridization with Tel G oligo probe also reveals telomere signals on all chromosome ends. Loading equal amounts of DNA in each lane is essential for determining the telomere terminal nucleotide by the adapter ligation assay and this is shown by th dium bromide staining of the gel in this protocol, the end labeled unique oligo and guide oligo adapters are only ligated to chromosome ends with the telomere g overhang sequence is compatible with a guide oligo.
Since the unique oligo is end labeled, the ligation product will be radioactive and give a strong signal. Lane one gives a strong signal indicating that a large amount of unique TG one adapter has been ligated to the telomere end. TG one has an ending sequence of C-C-C-T-A, hence the telomere g overhangs ligated to this adapter and is TT A GGG.
No significant amount of ligation product is observed using other guide oligos indicating that telomeres ending in TT A GGG are predominant in T brucey cells. Loading the end labeled guide oligo serves as a negative control and a size marker without ligation. The end labeled guide oligo is 22 nucleotides long.
The band at about 44 nucleotides is most likely the residue non denatured adapter after ligation to the chromosome end. The size of the extended product reflects the length of G overhang structure. Most T brucey telomere G overhangs end in TT A GGG.
However, these G overhangs appear to be very short at about 10 nucleotides. As products extended from the Ligated TG one guide oligo are not much longer than the guide oligo itself, although only a small amount of TG four adapter was ligated to the telomere ends products extended from ligated TG four are much longer with the longest product being about 55 nucleotides. Hence two types of telomere G overhang structures were observed in tbrc cells.
If the ligation indeed results from the telomere G overhang structure, treating the genomic DNA with XO one or XOT, which are three prime overhang specific nucleases should result in loss of the ligation products. Both types of G overhang structures are sensitive to such treatment. We've just shown you how to analyze the telomere giang structure in trapasso bru.
When doing this procedure, it's important to remember to load equal amount of samples in each lane. So that's it. Thanks for watching and good luck with your experiments.
