1994 Packard Instrument Company. IAN-007. Quantification of Double Strand Breaks in Mammalian. DNA Using Pulsed Field Gel Electrophoresis. Introduction.
IAN-007
Quantification of Double Strand Breaks in Mammalian DNA Using Pulsed Field Gel Electrophoresis
Introduction The study of the formation and repair of DNA double strand breaks (DSB’s) is an area of active research. These DNA lesions are thought to be important in chromosome aberrations, mutation induction, and cell death. Although other methods are available for measuring DSB’s (i.e., neutral elution, sucrose density gradients), pulsed field gel electrophoresis (PFGE) is being increasingly utilized.1 With PFGE, cells exposed to DSB-inducing agents such as radiation or chemicals are embedded in agarose blocks (to prevent mechanical shear), lysed, and then digested with proteinase K to free the DNA from protein. Blocks are placed in the sample wells of an agarose gel and subjected to PFGE. To assay DSB’s, PFGE is performed using long pulse times (~ one hour) to permit DNA fragments of up to 10 Mbp to migrate. Intact mammalian chromosomal DNA, however, is too large to migrate out of the sample block. After exposure to an agent that induces DSB’s, the relatively large DNA fragments migrate at a rate independent of size, and form a single band below the sample well. Very large radiation doses, which induce many DSB’s, produce fragments small enough to migrate at rates slightly dependent on size, and the peak may be shifted to a slightly higher mobility.1 The DNA in the band of fragments that migrates from the sample block can be quantified to obtain an estimate of DSB’s. The results are usually expressed as the amount of DNA in the migrating band as a fraction of the sum of the total DNA in this band plus that remaining in the sample plug.1,2 In the experiments reported here, DSB’s were produced in cells prelabeled with [14C]-thymidine, and the InstantImager® Electronic Autoradiography System was used to image the labeled DNA in dried agarose gels and to quantify the DSB’s. In one experiment DSB’s were determined as a function of radiation dose. In a second experiment, cells were PAN0055 1/94 Printed in U.S.A.
treated with the radiomimetic drug bleomycin, and in some cases DNA repair was allowed to occur before analysis. The InstantImager results from one of the experiments were compared with results obtained using liquid scintillation counting (LSC) of the bands from the same gel.
Methods Cells were labeled by incubation with 0.02 µCi/mL [ 14 C]-thymidine (1.85-2.22 GBq/mmol, ICN Biochemicals) for 20 hours in complete F10 medium. The cells were chased in nonradioactive medium for four hours before drug or radiation treatment to allow small pieces of DNA undergoing replication to become full size. Labeled wild-type V79 Chinese hamster cells were irradiated with from zero to 50 Gray of gamma rays before PFGE and analysis with the InstantImager and LSC. Multidrug resistant LZ-100 hamster cells were treated with 100 µg/mL of bleomycin for one hour, and then the drug was removed, and the cells were rinsed with fresh F12 medium. Some of these cells were incubated for one or two hours in fresh complete medium to allow DNA repair before analysis. Following irradiation or drug treatment, 1.5 X 106 cells were embedded in 2% Sea PlaqueTM agarose (FMC® BioProducts) in a precooled block former. After the agarose solidified, the cells were lysed and digested in the block with 1 mL of 2% Sarkosyl, 500 mM EDTA, 100 µg/mL proteinase K for 21 hours. The blocks were rinsed in TE buffer and sealed into sample wells in a 1% ultra pure agarose gel for PFGE. The gel electrophoresis was performed in a BioRad CHEF DR II apparatus for 15 hours at 25 oC at 40V with a switching time of 75 minutes.1 After electrophoresis, gels were stained with 0.5 µg/mL ethidium bromide for 20 minutes and photographed using UV transillumination. Gels were then dried at 50 to 60 oC for one to two hours in a SavantTM gel drier, and then immediately imaged © 1994 Packard Instrument Company
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and analyzed with the InstantImager. In one experiment the bands were cut out of the dried agarose gel with the aid of UV transillumination, placed in 100 µl of 10N HCl, heated briefly on a hot plate, mixed with 10 mL of LSC cocktail, and then counted using a Packard 2500TR LSC.
After imaging the dried gel with the InstantImager, the gel was placed on a UV transilluminator, the bands were cut out, and the amount of radioactivity measured using LSC. Essentially the same results were obtained with both instruments (Figure 4). DNA Repair After Drug Treatment Multidrug resistant (LZ-100) Chinese hamster cells prelabeled with [14C]-thymidine were exposed to 100 µg/mL bleomycin for one hour. A control aliquot of cells was incubated without the drug. Some of the cells were incubated after the drug treatment for either one or two hours to allow the cells to repair before blocks were made. The cellular DNA was then separated by PFGE, and the gel was stained with ethidium bromide and dried (Figure 5).
Results Radiation Dose-Response Chinese hamster cells prelabeled with [14C]-thymidine were irradiated with varying doses of gamma radiation (0, 3, 6, 12, 24, and 50 Gray), separated by PFGE and stained with ethidium bromide (Figure 1). The gel was then dried and imaged with the InstantImager for ten minutes (Figure 2). Lane one contained DNA from control cells that were not irradiated. Lanes two to six contained DNA from cells irradiated with increasing radiation doses (see Figures 3 and 4).
The dried gel was imaged with the InstantImager for 90 minutes (Figure 6). A template of four lanes (each 11 mm wide) was overlaid on the image, and profiles were created of all lanes. The areas under the peaks were integrated simultaneously in all four lanes with the profile region tool. Background regions were created near the bottom of each lane. Approximately 2000 net counts were counted in each lane.
A template of six lanes each 12.5 mm wide was overlaid on the image and a rectangular region of interest was created around one band and then copied around all the other bands. The rectangular regions of interest at the top of each lane represent background regions. The InstantImager software automatically assigned the regions to the correct lane according to the band positions. The software calculated the net counts in the regions of interest and also reported the counts as the percentage of the sum of net activity in the two bands in each lane (Figure 3).
The counts in the bands in each of the lanes were reported as the percentage of sum of the net counts in the two bands in each lane, and the report was exported to an Excel® spreadsheet using the Windows Clipboard feature. The results were then presented in a bar graph created using Excel (Figure 7).
Conclusion DNA labeled with 14C was imaged and quantitated with the InstantImager in dried agarose gels with acquisition times of ten or 90 minutes. The fraction of DNA that migrated out of the sample block was easily determined by placing rectangular regions of interest over the bands or by integrating the areas under profiles of lanes. Background was subtracted and the results calculated as a fraction of the sum of
Figure 1. Ethidium bromide fluorescence of DNA from irradiated Chinese hamster cells separated by PFGE.
Figure 2. Image of 14C-labeled DNA from irradiated Chinese hamster cells separated by PFGE. The image was acquired with the InstantImager in ten minutes. The template used to quantitate the DNA in each lane is shown in red. The rectangular regions of interest used to quantitate the DNA bands and the background in each lane are shown with marks at the centroid (center of gravity) positions of the counts in each region.
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Figure 3. The report produced by the InstantImager from the image in Figure 1. The Net Counts and the Net % Total (percentage of the sum of all regions in the lane) are reported in the last two columns for the bands in each lane. The Net % Total of band 1 in each lane provides a measure of the DSB’s in the DNA.
Figure 4. Double stranded breaks (DSB’s) in DNA irradiated with varying doses of gamma radiation expressed as the fraction of the DNA that migrated from the sample blocks. The results from the InstantImager (red circles) are shown with the results from the same gel counted by LSC (blue circles).
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Acknowledgment
the net counts in each lane of the gel. The results from the InstantImager correlated well with results obtained with LSC. Thus the InstantImager facilitates and speeds up the analysis of radioactively labeled PFGE samples.
We thank Dr. Marguerite Sognier of the University of Texas Medical Branch, Galveston, Texas for the opportunity to analyze the samples prepared in her laboratory and for many helpful suggestions.
References 1. Blöcher, D., Einspenner, M. and Zajackowski, J. (1989) CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks. Int. J. Radiat. Biol. 56: 437-448. 2. Stamato, T.D. and Denko, N. (1990) Asymmetric Field Inversion Gel Electrophoresis: A New Method for Detecting DNA Double-Strand Breaks in Mammalian Cells. Radiation Research 121: 196-205.
Figure 5. Ethidium bromide fluorescence of DNA from hamster cells treated with bleomycin and separated by PFGE.
Excel is a registered trademark of Microsoft Corporation. FMC is a registered trademark of FMC Corporation. Savant is a registered trademark of Savant Instruments, Inc.
Figure 6. Image of 14C-labeled DNA from bleomycin treated hamster cells separated by PFGE. The image was acquired with the InstantImager in 90 minutes. The template used to quantitate the DNA in each lane is shown in red. The profiles of all the lanes are shown in the box at the right. The profile of any single lane could be displayed by selecting the lane. The profile regions used to integrate the counts in the bands or to sample the background are drawn in each lane.
Figure 7. Excel chart showing the double stranded breaks caused in multidrug resistant hamster cells by bleomycin and the amount of DNA repair in one or two hours.
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