Markus Löbrich*, Björn Rydberg and Priscilla K. Cooper. Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA ...
1996 Oxford University Press
1802–1808 Nucleic Acids Research, 1996, Vol. 24, No. 10
Random-breakage mapping method applied to human DNA sequences Markus Löbrich*, Björn Rydberg and Priscilla K. Cooper Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA Received March 15, 1996; Accepted April 2, 1996
ABSTRACT The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453–4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated cells, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method. INTRODUCTION Techniques most commonly used for mapping of the human genome include random clone fingerprinting (1–3), hybridization fingerprinting with oligonucleotides (4) or selected classes of representative sequences (5), and fluorescence in situ hybridization (6,7). The smallest human chromosome, chromosome 21 (8), with a size of ∼50 megabasepairs (Mb) has been studied extensively (9–11) and has often represented a model for physical mapping of the whole human genome (12–14). The use of linking clones which enabled the localization of human disease-related genes (15,16) greatly facilitated the construction of a chromosome 21 physical map in recent works (17,18). However, methods which directly determine the position of a given
sequence inside a chromosome or fragment are often very complex (19,20) or require that the sequence contains a useful restriction site, as with the RecA-assisted restriction endonuclease (RARE) cleavage technique (21). A possible alternative approach is the random-breakage mapping method developed by Game et al. (22) and applied by them to yeast sequences. The distance between a cloned sequence and the ends of the molecule containing the sequence is determined in this approach by hybridization to molecules broken randomly by ionizing radiation and separated according to size by gel electrophoresis. The smear pattern below the band of unbroken molecules has two abrupt changes in intensity which reveal information about the location of the hybridizing sequence within the DNA molecule. The positions of the discontinuities correspond to the distance of the hybridizing sequence to the proximal and distal ends of the unbroken molecule. For a complete theoretical treatment, see Game et al. (22). We here demonstrate the applicability of this technique to mammalian genomic DNA by using the randombreakage mapping method to confirm the known position of a sequence on the human X chromosome (23). We have further applied this approach to determine the positions of three hybridization sequences on human chromosome 21 with respect to the ends of their corresponding NotI restriction fragments. One of these has been reported to have two possible locations on chromosome 21 (9,24), although it previously had been shown to be linked to another of the three sequences investigated here (12,25–27). The similar smear pattern of the two sequences absolutely excludes the second location and removes this uncertainty. This de novo application of the random-breakage mapping method to human DNA sequences demonstrates the utility of this approach. MATERIALS AND METHODS Cell culture Primary human skin fibroblasts GM38 from a normal individual were obtained from the NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ. Cell cultures were grown in a humidified 5% CO2 atmosphere at 37C in McCoy’s 5A medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO), 2 mM glutamine, 10 mM HEPES and antibiotic-
* To whom correspondence should be addressed at present address: Strahlenzentrum, Justus-Liebig-Universität Gießen, Leihgesterner Weg 217, 35392 Gießen, Germany
1803 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.110 Nucleic antimycotic solution (GIBCO). Confluent cell cultures were used in all experiments. Radiation sources X-irradiations were performed using a Philips Medical Systems RT 250 X-ray unit operated at 225 kVp, 15 mA with 0.35 Cu filtration. Dose rates were 1–2 Gy/min as determined with a Victoreen dosimeter. The Ne ion exposures were performed at the BEVALAC accelerator at Lawrence Berkeley Laboratory. Dose rates were in the range of 1–5 Gy/min. For further details, see Rydberg et al. (28). DNA preparation Cultures in 150 cm2 flasks containing ∼5 × 106 cells were harvested by trypsinization, resuspended at a concentration of 2 × 107 cells/ml in 0.6% low-melting-temperature agarose (Sigma, type VII) in phosphate-buffered saline (PBS) and cast into plug molds (20 × 9 × 1.2 mm). After irradiation of the plugs either with X-rays or, in some cases, with accelerated Ne ions, the embedded cells were lysed, digested with proteinase K, and the DNA restricted with NotI [for details, see Löbrich et al. (29)]. Restriction digestion conditions were optimized in a series of preliminary experiments, and neither higher concentrations of NotI nor longer incubation times changed the distribution of restriction fragments as given by either the ethidium bromide fluorescence or the hybridization pattern. Under these conditions restriction is essentially complete, since the proportion of the hybridization signal appearing above the band of unbroken restriction fragments was never more than 5% of the total signal for unirradiated controls and showed no dose-dependent changes. Pulsed-field gel electrophoresis The electrophoresis was performed with a CHEF DR II apparatus (BioRad) with a hexagonal array of 24 electrodes, which produce a field reorientation angle of 120. The plugs were inserted into 0.8% gels made from Ultrapure DNA Grade agarose (BioRad) in 0.5× TBE (1× TBE is 89 mM Tris, 89 mM boric acid and 2 mM Na2EDTA, pH 8). The gels were run at ∼12C in 0.5× TBE. Details of the running conditions for each experiment are given in the figure legends. The buffer was recirculated through a refrigeration unit to keep the temperature constant and to avoid ion build-up at the electrodes. Intact chromosomes of Schizosaccharomyces pombe and Saccharomyces cerevisiae (BioRad) were used as size standards. Southern hybridization Vacuum blotting of the gel was performed according to the manufactorer’s instructions (LKB 2016 VacuGene, Vacuum Blotting System, Pharmacia LKB Biotechnology AB, Sweden). Briefly, the DNA was partly depurinated by treating the gel with HCl, denatured with NaCl and neutralized. Transfer to a nylon reinforced nitrocellulose membrane (Hybond-C Extra, Amersham) was with SSC as transfer buffer. After blotting, the membrane was washed for 15 min in 5× SSC, air-dried and baked for 1 h at 80C. Hybridization was performed for 48 h at 42C in 10 ml 50% formamide, 6× SSPE (20× SSPE is 3 M NaCl, 0.2 M NaH2PO4, 20 mM Na2EDTA, pH 7.4), 1× Denhardt’s solution, 0.3 mg/ml
1803
denatured salmon sperm DNA, and 0.5% SDS containing ∼25 ng labeled DNA probe, basically according to Sambrook et al. (30). After hybridization the filters were washed three times for at least 1 h each in 2× SSPE, 0.1% SDS at room temperature, 30 min in 0.1× SSPE, 0.1% SDS at room temperature and again three times for at least 30 min each in 0.1× SSPE, 0.1% SDS at 65C. At least 200 ml washing solution were used per filter and washing step. Filters could be stripped and re-hybridized. DNA probes The DNA probes used were D21S1 (pPW228C, ref. 31), D21S11 (pPW236B, ref. 31) and D21S15 (pGSE8, ref. 32) for chromosome 21 and DXS1327 (pUC19, ref. 23) for the X chromosome. Each of these probes is known to hybridize to a single copy sequence. The NotI restriction fragment containing the sequences for the probes D21S1 and D21S11 has a size of 3.2 Mb in some cell lines including the one used in this work (33) and the fragment for the probe D21S15, which is located more distally on the same arm of chromosome 21 (at q22.3), is reported to have a size of 1.5 Mb (32). The probe DXS1327 has been shown to hybridize to a 3 Mb NotI restriction fragment on the human X chromosome and its location has been mapped (23). The plasmids were grown in appropriate Escherichia coli strains and isolated by standard procedures. Probes were prepared by cutting the insert human DNA from the vector with EcoRI and gel purifying them (30). Labeling of the probes with 32P was performed by the use of random priming essentially according to the manufacturer’s instructions (Amersham). This resulted in specific activities greater than 4 × 109 dpm/µg. Signal detection and analysis After washing, the membranes were wrapped in plastic wrap and used to expose X-ray film (Kodak XAR-5) with intensifying screens (Dupont Cronex Lightning-plus) at –70C for typically 2–4 days. Alternatively, analysis was performed using a phosphor-imaging-system (Molecular Dynamics, Model 400A). Exposure was usually overnight and image analysis was performed with a PC connected to the imaging system and with software provided by the manufacturer. RESULTS Principles of the method In this work we apply the random-breakage mapping method to determine the positions of three DNA sequences from human chromosome 21 with respect to the ends of their corresponding NotI fragments, and we verify the position of an X chromosome sequence previously determined by other methods (23). The random-breakage method allows the determination of the distance between a cloned sequence and the ends of the DNA molecule containing it. However, an ambiguity with regard to the two ends of the molecule exists and cannot be resolved by this method. A theoretical description of the random-breakage mapping method has been given in great detail elsewhere (22,34) and therefore is not treated comprehensively here. The basic idea of the method is best understood by considering DNA molecules which are randomly broken exactly once. The probability of breakage at a particular nucleotide site is assumed to be independent of the position inside the molecule and is therefore 1/N for a molecule consisting of N nucleotides. The result of the
1804 Nucleic Acids Research, 1996, Vol. 24, No. 10
Figure 1. Illustration of a DNA fragment of length N containing a hybridization site (black square) at a distance n from the closest end of the fragment.
break will be two smaller molecules, only one of which will contain the sequence for hybridization and will therefore be detectable. In addition, the hybridization signal is independent of the size of this fragment. These two features are in contrast to methods in which all molecules are detected (e.g. by staining with ethidium bromide), with each molecule contributing to the overall signal in proportion to its size. In the case of exactly one break, the molecule with the hybridization site will have a minimum size of n, which represents the distance of the hybridization site to the proximal end of the original molecule (Fig. 1). Since at least two breaks would be necessary to produce smaller molecules, these are much less probable if the majority of the molecules have either no or one break, and therefore a marked decrease in the hybridization intensity at the position corresponding to size n exists. The probability of producing a fragment of size n or larger equals 1/N up to a second critical size N–n, representing the distance between the hybridization point and the distal end of the original molecule (again for molecules broken exactly once). The probability for sizes larger than N–n is 2/N, twice as great as the probability of producing molecules between n and N–n. This is due to the fact that a break close to either of the two ends of the original molecule will produce such a size while a particular size between n and N–n can be produced at only one position. There is, therefore, a second change in the hybridization intensity at the position corresponding to size N–n. For the case in which two breaks are produced inside the molecule the argument is similar, though more complicated, and also predicts two sharp intensity changes at the positions n and N–n. These changes are most easily detected for one break per molecule and decrease in sharpness with increasing numbers of breaks. For this reason, experiments employing the random-breakage mapping method should be performed under conditions in which oncebroken molecules predominate.
Application of the method to probe D21S15 on a 1.2 Mb NotI restriction fragment Normal human fibroblasts were irradiated with X-rays in order to induce DNA breaks, lysed in agarose and digested with NotI restriction enzyme, then separated by means of a PFGE system according to size. Figure 2 (upper panel) shows an ethidium bromide stained PFGE gel. Lanes 1 and 2 contain yeast DNA length standards (S.pombe and S.cerevisiae, respectively) with sizes indicated in Mb on the left side of the picture. The third lane contains NotI restricted DNA from unirradiated cells, and lanes 4–10 contain DNA from cells exposed to increasing X-ray doses. Most of the DNA from unirradiated cells enters the gel after restriction, producing a distribution of NotI fragment sizes. This distribution is shifted to lower average molecular weight with increasing radiation dose due to induction of double-strand breaks (dsbs). Beneath the wells two compression zones (at the
Figure 2. Positioning of probe D21S15 within the NotI restriction fragment. Upper panel: ethidium bromide stained gel with NotI-restricted DNA from irradiated human fibroblasts separated by means of PFGE at a field strength of 6 V/cm for 16 h and linearly increasing pulse times from 50 to 150 s. Lanes 1 and 2, S.pombe and S.cerevisiae chromosomes (numbers on the left indicate sizes in Mb); lane 3, unirradiated DNA; lanes 4–10, DNA from cells irradiated with 40, 70, 100, 130, 160, 200 or 240 Gy of X-rays. Middle panel: Southern blot of the gel visualized on X-ray film. Lower panel: Computer-generated phosphor image of the same Southern blot. The top arrow in each panel shows the location of the NotI fragment containing the hybridization site for D21S15. The smear of broken NotI fragments below this band has two discontinuities (indicated by the two lower arrows) revealing the position of the hybridization site with respect to the ends of the restriction fragment.
position of the S.pombe chromosomes) show a peculiar behaviour. It can be seen that the lower of the two compression zones disappears faster with dose than the upper zone, suggesting that it contains larger NotI fragments. Similar inversion effects have been previously described (35). However, over the size-range of interest, indicated by the arrows on the right side of the picture, the separation is approximately linear with size as shown by the S.cerevisiae markers in lane 2. Previous studies by us (36) have ascertained that, with the electrophoresis conditions used, no separation artifacts (such as inversion) occur for these S.cerevisiae markers. The DNA in the gel was transferred to a membrane and hybridized with probe D21S15. The middle panel of Figure 2 is an X-ray film showing the hybridization signal with a single band in the unirradiated control DNA in lane 3 at an apparent size of
1805 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.110 Nucleic 1.3 Mb (upper arrow on the right side of the picture). The intensity of the band decreases with increasing radiation dose as dsbs induced in the restriction fragment produce smaller molecules, resulting in the appearance of a smear under the band. This smear shows the predicted feature: two sharp changes in intensity, indicated by the two lower arrows. These are especially clear in lanes 6–9. Based on our previously determined dsb induction rate for X-rays of 5.8 × 10–3 breaks/Mb/Gy (29), the 1.3 Mb restriction fragment received an average of 0.75–1.5 breaks for the radiation doses corresponding to these lanes (100–200 Gy), supporting the aforesaid principle of the method. The lower panel of Figure 2 is a computer-generated representation of the phosphorimage of the same filter as shown in the X-ray film. Since both show the same feature of two sharp intensity changes, the remote possibility that the intensity changes are an artifact of non-linear behaviour of X-ray films can be excluded. The band, as well as the two changes in intensity, are again marked by arrows and their corresponding positions in the ethidium bromide picture (upper panel) are also indicated. It is apparent that the two cut-offs disappear at the very high dose and that the lower cut-off appears at higher doses than the upper cut-off. This latter point is treated theoretically in the work of Game et al. (22) and is due to the fact that the signal above the upper cut-off arises from either of two possible break sites for any given point, so this signal is generated at lower doses than the signal just above the lower cut-off, where there is only one site per size position. The size determination of the lower cut-off is sufficient for mapping the hybridization sequence with respect to the ends of the NotI restriction fragment, but a control can be achieved with the additional size determination of the upper cut-off, because both sizes should add up to the size of the original NotI fragment. In several experiments similar to that of Figure 2 performed with different probes under various electrophoresis conditions, a higher value for the sum of the two discontinuities was found compared with the measured size of the unbroken fragment. The sum was usually ∼15–20% higher than the intact size. Since the migration behaviour of DNA in pulsed-field gels is known to depend strongly on the DNA concentration used (37), we performed experiments to determine the sizes of the restriction fragments and the discontinuities more accurately. Unirradiated DNA, electrophoresed in decreasing concentrations, was hybridized with probes recognizing the 1.3 Mb fragment and a 3.2 Mb fragment as described in the next section (data not shown). Also, membranes containing irradiated DNA at our usual working concentration (as described in Materials and Methods) and showing the smear pattern with the two discontinuities for a probe recognizing the 3.2 Mb fragment were stripped and re-hybridized with the probe recognizing the smaller NotI fragment. This allowed direct comparison of the sizes of the discontinuities with NotI fragment sizes and circumvented the need for size determination of the discontinuities at lower concentrations, when they are barely visible. As a result of these experiments for accurate size determinations we found the apparent sizes of the molecules at the concentration normally used to be bigger than their correct sizes determined at lower concentrations. In addition, this overestimation in size was found to depend strongly on the size of the molecules and the electrophoresis conditions used. For a fixed electrophoresis condition, the effect (expressed in percent overestimation) increased with decreasing molecular weight, in agreement with work of other authors (37). This explains why the sum of the two cut-offs determined from the experiment shown in Figure 2 and
1805
other similar experiments was usually 15–20% higher than the size of the original band. After taking the concentration effect into account, the size of the NotI fragment containing the hybridization site for D21S15 was determined to be 1.2 Mb, while the distance of the hybridization sequence to the nearest end of the fragment, corresponding to the position of the lower cut-off, was 0.4 ± 0.03 Mb. Mapping of D21S1 on a 3.2 Mb NotI restriction fragment Since the second probe being mapped hybridized to a 3.2 Mb NotI fragment, different electrophoresis conditions for size separation were applied for these studies. Figure 3A shows an ethidium bromide stained PFGE gel from an experiment in which cells were irradiated with accelerated Ne particles. The use of accelerated heavy ions in this experiment instead of X-rays was for reasons in connection with other ongoing projects and has no particular relevance to the mapping application described here. With regard to the random-breakage mapping method, any kind of radiation can be applied as long as DNA dsbs are produced randomly. The first lane contains S.pombe (left part) and S.cerevisiae (right part) chromosomes as length standards with sizes indicated in Mb on the left side of the picture. Lane 2 contains unirradiated but NotI digested DNA, and lanes 3–8 contain the irradiated DNA samples. Again, the distribution of NotI fragments shifts with increasing radiation dose to lower molecular weight. The X-ray film from the Southern blot of this gel is shown in Figure 3C and described below. Figure 3B is a computer-generated representation of the phosphorimage from hybridization of the probe D21S1 to a blot of a similar gel from a separate experiment, for which the electrophoresis conditions were identical to those of Figure 3A. Figure 3D shows the X-ray film corresponding to the phosphorimage in Figure 3B. Lanes 1 of Figure 3B and D, which contain unirradiated control DNA, show a single band at 3.2 Mb (indicated by the upper arrow). Lanes 2–6 contain DNA samples irradiated with increasing doses of accelerated Ne particles. The two intensity changes in the smear below the band are again indicated by the two lower arrows, and the computer-generated image in Figure 3B is aligned with the corresponding positions in the ethidium bromide stained gel of Figure 3A. The upper cut-off is most easily seen in lane 3 (40 Gy), while the lower cut-off is clearest for higher doses (lanes 5 and 6, 100 and 120 Gy). After taking the above mentioned concentration effect into account, the distance of the hybridization sequence for D21S1 to the closest end of the 3.2 Mb NotI fragment was determined from the position of the lower cut-off to be 1.2 Mb. Figure 3C shows an X-ray film obtained from hybridization of the gel shown in Figure 3A with the same probe as used previously in Figure 2, D21S15. Lane 2 contains unirradiated DNA, and lanes 3–8 contain DNA from cells irradiated with increasing doses of accelerated Ne particles. Again, the band representing the unbroken fragments as well as the two cut-offs are visible and indicated by arrows. Although the electrophoresis conditions applied in this case were optimized for the larger restriction fragment, the positions of the cut-offs are consistent with the results shown in Figure 2. In addition, since the DNA samples in Figure 3C and D were separated with the same electrophoresis conditions, it is possible to align the two pictures. The lower cut-off in the hybridization signal of D21S1 (Fig. 3D) appears at about the same position as the band for the intact NotI
1806 Nucleic Acids Research, 1996, Vol. 24, No. 10
Figure 3. Positioning of probes D21S1 and D21S15 within their respective NotI restriction fragment. (A) Ethidium bromide stained gel with NotI restricted DNA from irradiated human fibroblasts separated by means of PFGE at a field strength of 1.5 V/cm for 120 h and linearly increasing pulse times from 50 to 5000 s. Lane 1,S.pombe and S.cerevisiae chromosomes (numbers on the left indicate sizes in Mb); lane 2, unirradiated DNA; lanes 3–8, DNA from cells irradiated with 7, 20, 60, 100, 140 or 160 Gy of accelerated Ne particles. (B) Computer-generated phosphor image showing the hybridization signal of the probe D21S1 with the main band respresenting the unbroken fragments (upper arrow) and the two discontinuities in the smear below indicated by the two lower arrows. This Southern blot was derived from a gel prepared with the same electrophoresis conditions as the one in the upper left panel, and the hybridization signal is aligned to the corresponding position in the gel. Lane 1, unirradiated DNA; lanes 2–6, DNA from cells irradiated with 11, 40, 80, 100 or 120 Gy of accelerated Ne particles. (C) X-ray film showing the hybridization signal of the probe D21S15 with the main band and the two discontinuities in the smear below (indicated by the upper and the two lower arrows, respectively). This Southern blot was derived from the gel in (A) and is aligned to the blot in (D). (D) X-ray film visualizing the same hybridization signal shown in (B).
fragment containing the hybridization site for D21S15 (Fig. 3C), giving an independent estimate of this cut-off at 1.2 Mb.. It can be seen for the experiment of Figure 3 that even in the unirradiated samples a fraction of the hybridization signal appears below the band for unbroken fragments. This is probably due to limited degradation of unirradiated DNA. Since this background level of breaks corresponds to a low average break frequency, the hybridization signal from degraded DNA should in principle constitute mainly a smear above the upper cut-off position. However, the visible background seems to appear rather as a band at the position of the lower cut-off. Theoretically, such a band phenomenon would only be expected for a high break frequency (more than one break per fragment). This puzzling observation might be better understood by assuming that a subpopulation of the DNA from unirradiated cells is subjected to a significant degree of degradation (more than one break per fragment) while the majority of the cells show very little or no DNA degradation. This scenario would be in agreement with a background smear corresponding to a low average break frequency as well as the appearance of a band at the position of the lower cut-off. Mapping of D21S11 in relation to D21S1 A second probe D21S11 for the same 3.2 Mb NotI restriction fragment that contains D21S1 has been suggested to have two possible locations on chromosome 21 (9,24), although it had been previously reported to be close to D21S1 (12,25–27). One proposed location is close to a probe D21S110 which is located at a distance of 3.32 Mb from D21S1, and the other is close to D21S232 which is only 0.18 Mb apart from D21S1 (24). We performed random-breakage mapping experiments with the probes D21S1 and D21S11 to resolve this uncertainty and to test the utility of the described mapping approach. Figure 4 shows a computer-generated version of the phosphorimage of a mem-
brane hybridized with D21S11 (left panel) along with an image of another membrane hybridized with D21S1 (right panel). Both membranes were generated with the same electrophoresis conditions, identical to those of the experiments in Figure 3. Lane 1 in both panels contains unirradiated DNA, while the remaining lanes contain DNA of cells irradiated with increasing doses of X-rays. It can be seen that the two hybridization patterns are indistinguishable with respect to the positions of the cut-offs. Therefore, the two sequences must be positioned at the same distance from the ends of the restriction fragment (with the limit of resolution being ±0.1 Mb). Hence, they are either close to each other at a distance of 1.2 Mb from the same NotI end or separated by 0.8 Mb and positioned at a distance of 1.2 Mb from opposite NotI ends, since the intact NotI fragment has a size of 3.2 Mb. A previously published work (27) placing D21S11 within 100 kb of D21S1 is in agreement with the first of these possibilities. Our mapping result excludes the possibility that D21S11 is located close to D21S110. Application of the method to the previously mapped probe DXS1327 on a 3 Mb NotI restriction fragment The X chromosome probe DXS3127 has been mapped to a 3 Mb NotI restriction fragment containing hprt and assigned a location 210 kb telomeric of hprt, with an uncertainty range of 130–220 kb (23). This placement corresponds to a distance of ∼500–600 kb from the closest end of the NotI fragment, the end which is telomeric from hprt. We have applied the random-breakage mapping method to confirm this result and thus to demonstrate the validity of the technique. Figure 5 shows an X-ray film with a signal arising from the hybridization of DNA samples from X-irradiated cells with probe DXS1327. The DNA in lane 1 contains ∼0.4 breaks/restriction fragment, while the DNA in lane 2 contains ∼1.3 breaks/fragment. The upper arrow on the left
1807 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.110 Nucleic
Figure 4. Positioning of probes D21S11 and D21S1 within their NotI restriction fragment (both probes hybridize to the same NotI fragment). NotI-restricted DNA from irradiated human fibroblasts was separated by means of PFGE at a field strength of 1.5 V/cm for 120 h and linearly increasing pulse times from 50 to 5000 s. Left panel: computer-generated phosphorimage showing the hybridization signal of the probe D21S11 with the main band representing the unbroken fragments and the two discontinuities in the smear below. Lane 1, unirradiated DNA; lanes 2–5, DNA from cells irradiated with 20, 40, 60 or 80 Gy of X-rays. Right panel: computer-generated phosphorimage showing the hybridization signal of the probe D21S1 with the main band and the two discontinuities in the smear below. Lane 1, unirradiated DNA; lanes 2–6, DNA from cells irradiated with 10, 20, 40, 60 or 80 Gy of X-rays. The hybridization patterns of the two probes are indistinguishable, showing the discontinuities at the same positions.
Figure 5. Positioning of probe DXS1327 within the NotI restriction fragment. NotI-restricted DNA from X-irradiated human fibroblasts was separated by means of PFGE at a field strength of 1.5 V/cm for 120 h and linearly increasing pulse times from 50 to 5000 s. Lanes 1 and 2, DNA containing about 0.4 and 1.3 breaks/3 Mb, respectively. The intact 3 Mb NotI fragment and the discontinuities at 2.4 and 0.6 Mb are indicated by arrows on the left.
indicates the position of the intact NotI fragment, which we measure at 3 Mb in agreement with the previous report, and the two lower arrows point to the two cut-offs, which we determine to be at 2.4 and 0.6 Mb. The lower cut-off, which contains the important mapping information, is particularly well-defined in lane 2. The electrophoresis conditions applied for this experiment were identical to the conditions of the experiments in Figures 3 and 4. The limit of resolution for the positioning of the sequence for DXS1327 was 0.1 Mb. The random-breakage mapping method hence places DXS1327 at 500–700 kb from the closest end, in good agreement with the position previously determined by physical mapping using a variety of restriction enzymes and analysis of deletion mutations (23). DISCUSSION We have used the random-breakage mapping method developed by Game et al. (22) to confirm the known map position for a sequence in the vicinity of hprt on the X chromosome and to map three human DNA sequences from chromosome 21 with respect to the ends of their NotI restriction fragments. It is a de novo application to mammalian DNA because its use has previously only been demonstrated for yeast DNA (22). Since yeast cells have an ∼200 times smaller genome size and the target DNA for
1807
hybridization therefore represents a much higher fraction of the total DNA, the method was in that case greatly facilitated. In the present experiments we introduced random breakage in DNA by irradiation of intact cells before lysis. However, irradiation can also be carried out on isolated DNA after lysis (22). In some applications NotI-digested DNA in agarose plugs may already be available or can be prepared in bulk quantities. The use of the random-breakage mapping method will then only involve irradiation of such plugs with X-rays before running out the DNA on pulsed-field gels, followed by Southern blotting. It is also possible to map sequences directly on mammalian chromosomes without restriction of the DNA in the special case in which their distance to the chromosome end is smaller than the size limit of resolution of the PFGE used (usually ∼10 Mb), since the lower cut-off in that case is expected to be visible inside the gel. One potential problem with restriction of DNA at high concentrations within agarose plugs is incomplete digestion, which would produce additional bands at higher molecular weight. Radiation breakage of those larger DNA pieces could smear the upper cut-off arising from breakage of completely digested molecules. However, the lower cut-off is not expected to be smeared out and should still provide the mapping information, as long as enough molecules are completely digested to produce a sufficient hybridization signal at this position. Other possible problems with the method include the potential for appearance of minor bands within the smear due to cross-hybridization to similar sequences elsewhere or partial digestion of internal, predominantly methylated restriction sites on the same fragment. Confusion of such possible hybridization signals with genuine cut-offs in the distribution can be prevented by accurately determining the sizes of both apparent cut-offs to verify that the sum agrees with the size of the intact fragment. However, the position of the lower cut-off alone is sufficient for mapping, and it is both more easily seen than the upper cut-off and less likely to be confused with minor bands from other sources. The effect of the DNA concentration on mobility must also be taken into account. If DNA concentrations >50 µg/ml are necessary to obtain sufficient hybridization signal for evaluation, experiments for size corrections should be performed. It is useful for such experiments to employ probes which hybridize to fragments of known sizes and which themselves are subject for mapping, as was the case in the present work. A systematic description of the concentration effect can be found in the work of Doggett et al. (37). The limit of resolution for this technique is not an absolute number but rather is dependent on the size of the molecules containing the hybridization sequence. In the experiments reported here the limit is estimated to be 0.1 and 0.03 Mb for the probes positioned on the 3 and 3.2 Mb fragments and on the 1.2 Mb fragment, respectively, which corresponds to an uncertainty in the determination of the migration distance and therefore the size of ±3%. The technique can be extended to larger or smaller fragments. If the fragment is too large to enter the gel, analysis is still possible by the use of separation conditions that are suitable for the size range of the lower cut-off. In addition, the use of constant field gel electrophoresis and very high radiation doses should allow mapping of sequences on DNA fragments as small as a few kb. However, for very small fragments the size of the DNA probe itself can be important and in that case represents the limit of resolution, since the probe is assumed in the theoretical treatment to be a point on the DNA molecule.
1808 Nucleic Acids Research, 1996, Vol. 24, No. 10 The utility of the random-breakage mapping method is demonstrated by the experiment shown in Figure 4, which eliminates one of two possible locations for the probe D21S11 by confirming its close proximity to D21S1. Even in the case of several different proposed locations for a probe on a particular restriction fragment, the random-breakage mapping method will often eliminate all possible locations but one, although by itself it determines the location only with respect to the ends of the molecule. The validity of the technique has been demonstrated by the experiment shown in Figure 5. The position of the probe DXS1327 on a 3 Mb NotI fragment, which had been previously determined by different methods (23), was confirmed by random-breakage mapping. ACKNOWLEDGMENTS We are very grateful to John Game for critically reading the manuscript and for many helpful discussions. We thank Ely Kwoh for help in preparing the figures. This work was supported by Order No. W-18265 from the US National Aeronautics and Space Administration to the NSCORT in Radiation Health at the Lawrence Berkeley National Laboratory and by the Office of Energy Research, Office of Health and Environmental Research, Health Effects Research Division, US Department of Energy, under Contract No. DE-AC03-76SF00098.
REFERENCES 1 Olson, M.V., Dutchik, J.E., Graham, M.Y., Brodeur, G.M., Helms, C., Frank, M., MacCollin, M., Scheinman, R. and Frank, T. (1986) Proc. Natl. Acad. Sci. USA 83. 7826–7830. 2 Kohara, Y., Akiyama, K. and Isono, K. (1987) Cell 50, 495–508. 3 Daniels, D.L., Olson, C.H., Brumley, R. and Blattner, F.R. (1990) Nucleic Acids Res. 18, 1312. 4 Craig, A.G., Nizetic, D., Hoheisel, J.D., Zehetner, G. and Lehrach, H. (1990) Nucleic Acids Res. 18, 2653- 2660. 5 Stallings, R.L., Torney, D.C., Hildebrand, C.E., Longmire, J.L., Deaven, L.L., Jett, J.H., Dogget, N.A. and Moyzis, R.K. (1990) Proc. Natl. Acad. Sci. USA 87, 6218–6222. 6 Fan, Y.-S., Davis, L.M. and Shows, T.B. (1990) Proc. Natl. Acad. Sci. USA 87, 6223–6227. 7 Van den Engh, G., Sachs, R. and Trask, B.J. (1992) Science 257, 1410–1412. 8 Korenberg, J.L. and Engels, W.R. (1978) Proc. Natl. Acad. Sci. USA 75, 3382–3386. 9 Chumakov, I., Rigault, P., Guillou, S., Ougen, P., Billaut, A., Guasconi, G., Gervy, P., LeGall, I., Soularue, P., Grinas, L. et al. (1992) Nature 359, 380–387.
10 Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L. et al. (1991) Nature 349, 704–706. 11 Lehesjoki, A.-E., Koskiniemi, M., Sistonen, P., Miao, J., Hästbacka, J., Norio, R. and de la Chapelle, A. (1991) Proc. Natl. Acad. Sci. USA 88, 3696–3699. 12 Gardiner, K., Horisberger, M., Kraus, J., Tantravahi, U., Korenberg, J., Rao, V., Reddy, S. and Patterson, D. (1990) EMBO J. 9, 25–34. 13 Cox, D.R., Burmeister, M., Price, E.R., Kim, S. and Myers, R.M. (1990) Science 250, 245–250. 14 Burmeister, M., Kim, S., Price, E.R., de Lange, T., Tantravahi, U., Myers, R.M. and Cox, D.R. (1991) Genomics 9, 19–30. 15 Fountain, J.W., Wallace, M.R., Bruce, M.A., Seizinger, B.R., Menon, A.G., Gusella, J.F., Michels, V.V., Schmidt, M.A., Dewald, G.W. and Collins, F.S. (1989) Science 244, 1085–1087. 16 Borrow, J., Goddard, A.D., Sheer, D. and Solomon, E. (1990) Science 249, 1577–1580. 17 Ichikawa, H., Hosoda, F., Arai, Y., Shimizu, K., Ohira, M. and Ohki, M. (1993) Nature Genet. 4, 361–365. 18 Wang, D. and Smith, C.L. (1994) Genomics 20, 441–451. 19 Pavan, W.J., Hieter, P. and Reeves, R.H. (1990) Proc. Natl. Acad. Sci. USA 87, 1300–1304. 20 Smith, C.L. and Condemine, G. (1990) J. Bacteriol. 172, 1167–1172. 21 Ferrin, L.J. and Camerini-Otero, R.D. (1991) Science 254, 1494–1497. 22 Game, J.C., Bell, M., King, J.S. and Mortimer, R.K. (1990) Nucleic Acids Res. 18, 4453–4461. 23 Lippert, M.J., Albertini, R.J. and Nicklas, J.A. (1995) Mutat. Res. 326, 39–49. 24 Lawrence, S., Collins, A., Keats, B.J., Hulten, M. and Morton, N.E. (1993) Proc. Natl. Acad. Sci. USA 90, 7210–7214. 25 Tanzi, R.E., Haines, J.L., Watkins, P.C., Stewart, G.D., Wallace, M.R., Hallewell, R., Wong, C., Wexler, N.S., Conneally, P.M. and Gusella, J.F. (1988) Genomics 3, 129–136. 26 Petersen, M.B., Slaugenhaupt, S.A., Lewis, J.G., Warren, A.C., Chakravarti, A. and Antonarakis, S.E. (1991) Genomics 9, 407–419. 27 Tassone, F., Cheng, S. and Gardiner, K. (1992) Am. J. Hum. Genet. 51, 1251–1264. 28 Rydberg, B., Löbrich, M. and Cooper, P.K. (1994) Radiat. Res. 139, 133–141. 29 Löbrich, M., Rydberg, B. and Cooper, P.K. (1994) Radiat. Res. 139, 142–151. 30 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 31 Watkins, P.C., Tanzi, R.E., Gibbons, K.T., Tricoli, J.V., Landes, G., Eddy, R., Shows, T.B. and Gusella, J.F. (1985) Nucleic Acids Res. 13, 6075–6088. 32 Wang, D., Fang, H., Cantor, C.R. and Smith, C.L. (1992) Proc. Natl. Acad. Sci. USA 89, 3222–3226. 33 Ichikawa, H., Shimizu, K., Saito, A., Wang, D., Oliva, R., Kobayashi, H., Kaneko, Y., Miyoshi, H., Smith, C.L., Cantor, C.R. and Ohki, M. (1992) Proc. Natl. Acad. Sci. USA 89, 23–27. 34 Cook, V.E. and Mortimer, R.K. (1991) Radiat. Res. 125, 102–106. 35 Chu, G. (1991) Proc. Natl Acad. Sci. USA 88, 11071–11075. 36 Löbrich, M., Ikpeme, S. and Kiefer, J. (1993) Anal. Biochem. 208, 65–73. 37 Doggett, N.A., Smith, C.L. and Cantor, C.R. (1992) Nucleic Acids Res. 20, 859–864.
