Asymmetric Field Inversion Gel Electrophoresis Research Articles

The contribution of thermally labile sugar lesions to DNA double-strand break formation in cells grown in the presence of BrdU

Purpose: Radiosensitization by bromodeoxyuridine (BrdU) is commonly attributed to an increase in the yield of double-strand breaks (DSB) in the DNA and an associated decrease in the reparability of these lesions. Radiation chemistry provides a mechanism for the increased yield of DSB through the generation, after bromine loss, of a highly reactive uracilyl radical that attacks the sugar moiety of the nucleotide to produce a single-strand break (SSB). The effects underpinning DSB repair inhibition remain, in contrast, incompletely characterized. A possible source of reduced reparability is a change in the nature or complexity of the DSB in BrdU-substituted DNA. Recent studies show that DSB-complexity or DSB-nature may also be affected by the presence within the cluster of thermally labile sugar lesions (TLSL) that break the DNA backbone only if they chemically evolve to SSB, a process thought to occur within the first hour post-irradiation. Since BrdU radiosensitization might be associated with increased yields and reduced reparability of DSB, we investigated whether BrdU underpins these effects by shifting the balance in the generation of TLSL.Methods and materials: We employed asymmetric-field-inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis (PFGE) method to quantitate DSB in a battery of five cells lines grown in the presence of different concentrations of BrdU. We measured specifically the yields of promptly forming DSB (prDSB) using low temperature lysis protocols, and the yields of total DSB (tDSB = prDSB + tlDSB; tlDSB form after evolution to SSB of TLSL) using high temperature lysis protocols.Results: We report that incorporation of BrdU generates similar increases in the formation of tlDSB and prDSB, but variations are noted among the different cell lines tested.Conclusions: The similar increase in the yields of tlDSB and prDSB in BrdU substituted DNA showed that shifts in the yields of these forms of lesions could not be invoked to explain BrdU radiosensitization.

Radiation Induced DNA Double Strand Break Studies of a Metal Sensitive Novel Bacterial Isolate from East Calcutta Wetland

Problem statement: This study was an attempt to isolate anaerobic microbes with potential for DNA double strand break repair using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. It also intended to verify the specificity of the medium for isolation of the desired family of microbe. Approach: Culture based technique was used to obtain the pure isolate that was further characterized in details. For double strand break repair studies, isolate was irradiated with different doses of 60Co gamma rays and its subsequent repair was observed using pulse field gel electrophoresis and asymmetric field inversion gel electrophoresis. Inhibitor was used to predict the mechanism of repair. Results: In this study we isolated and characterized a metal sensitive anaerobic microbial strain obtained using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. The strain was one of the members of the group of uncultivated bacterium as evident from phylogenetic analysis, thus indicating the successful cultivation of an as yet uncultivable novel microbe (GenBank Acc. No. FJ 930097) and also the non-specific growth of microbes in prescribed medium. It was a Gram positive Bacilli, member of Fermicutes with optimum growth at 25°C and pH-7. The growth curve analysis showed a lag phase up to 24 h, log phase from 24-48 h, an early stationary phase from 96 h onwards. The strain could repair the DNA double strand break caused by irradiation with 60Co γ rays. The dose profile study revealed maximum repair at 60 Grays and thereafter a drop in repair ability with increase in irradiation dose. The time required for repair showed an essential incubation period of 4 h. The DNA polymerase inhibitor, Arabinose CTP inhibited the repair indicating the involvement of polymerase in the repair process and thus pointing towards homologous recombination as the underlying mechanism. Conclusion: In this study we were able to cultivate an as yet uncultivable anaerobic bacterial isolate and predict the growth conditions for the isolate. On irradiation with 60Co γ rays the isolate showed maximum repair following 60 Gray damage. DNA polymerase inhibitor arabinose CTP inhibited the repair mechanism completely. This indicated that DNA polymerase took active part in repair process and thus the mechanism was that of homologous recombination repair.

DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma hepG2 cell line.

To investigate the role of DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma HepG(2) cell lines. Hep G(2) cells were exposed to hyperthermia and irradiation. Hyperthermia was given at 45.5 degrees C. Cell survival was determined by an in vitro clonogenic assay for the cells treated with or without hyperthermia at various time points. DNA DSB rejoining was measured using asymmetric field inversion gel electrophoresis (AFIGE). The DNA-PKcs activities were measured using DNA-PKcs enzyme assay system. Hyperthermia can significantly enhance irradiation-killing cells. Thermal enhancement ratio as calculated at 10 % survival was 2.02. The difference in radiosensitivity between two treatment modes manifested as a difference in the alpha components and the almost same beta components, which alpha value was considerably higher in the cells of combined radiation and hyperthermia as compared with irradiating cells (1.07 Gy(-1) versus 0.44 Gy(-1)). Survival fraction showed 1 logarithm increase after an 8-hour interval between heat and irradiation, whereas DNA-PKcs activity did not show any recovery. The cells were exposed to heat 5 minutes only, DNA-PKcs activity was inhibited at the nadir, even though the exposure time was lengthened. Whereas the ability of DNA DSB rejoining was inhibited with the increase of the length of hyperthermic time. The repair kinetics of DNA DSB rejoining after treatment with Wortmannin is different from the hyperthermic group due to the striking high slow rejoining component. Determination with the cell extracts and the peptide phosphorylation assay, DNA-PKcs activity was inactivated by heat treatment at 45.5 degrees C, and could not restore. Cell survival is not associated with the DNA-PKcs inactivity after heat. DNA-PKcs is not a unique factor affecting the DNA DSB repair. This suggests that DNA-PKcs do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.

Rejoining of DNA Double-Strand Breaks in Ku80-Deficient Mouse Fibroblasts

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

Induction of DNA Double-Strand Breaks by Restriction Enzymes in X-Ray-Sensitive Mutant Chinese Hamster Ovary Cells Measured by Pulsed-Field Gel Electrophoresis

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.

Dependence of fluorodeoxyuridine-mediated radiosensitization on S phase progression.

Recent evidence casts doubt on the hypotheses that fluoropyrimidine-mediated radiosensitization is related to cytotoxicity or to cell cycle redistribution into the G1/S boundary. We hypothesized that cells that are capable of progressing into S phase in the presence of fluorodeoxyuridine may also be more susceptible to radiation-induced damage. To test this hypothesis, fluorodeoxyuridine (FdUrd)-treated HT29 human colon cancer cells were separated by centrifugal elutriation into four fractions (1-4) containing a range of cells from those at the G1/S boundary (fraction 1) to those which had progressed approximately 11% into S phase (fraction 4). We found that fraction 4 cells showed significantly greater radiosensitization than fraction 1 cells. We also compared the effects of fluorodeoxyuridine on HT29 and SW620 human colon cancer cells. We found that, in contrast with HT29 cells, SW620 cells arrested at the G1/S boundary and were minimally radiosensitized. Finally, we found that an increase in sensitivity was correlated with a decrease in the rate of repair of DNA double-strand and single-strand breaks (assessed by asymmetric field inversion gel electrophoresis and alkaline elution respectively). These findings are consistent with the hypothesis that fluorodeoxyuridine-mediated radiosensitization depends on S phase progression and a decreased ability to repair radiation-induced DNA damage.

Two Methods for Assaying DNA Double-Strand Break Repair in Mammalian Cells by Asymmetric Field Inversion Gel Electrophoresis

The rejoining of gamma-ray-induced DNA double-strand breaks (DSBs) in mammalian cells was measured after various doses of gamma rays by using a version of pulsed-field gel electrophoresis to elute fragments of DNA from an agarose plug into the lane of an agarose gel. Two approaches for measuring the kinetics of DNA repair were compared. In the first method, cells are irradiated and incubated at 37 degrees C in monolayers, after which the cells are suspended in agarose and DNA is isolated and subjected to electrophoresis. In the second approach, cells are suspended in agarose first, then irradiated and incubated for repair, and the DNA is isolated for electrophoresis. In both methods the kinetics of repair appears to be biphasic, with an initial fast phase and a second slow phase. At equal doses the t1/2 for fast repair is two-fold less in cells incubated in monolayers than in cells suspended in agarose (11 min compared to 20-23 min) and threefold less after subtracting the slow repair component. In the agarose method the t1/2 values for fast repair increase with increasing radiation dose, while in the monolayer method they are constant. In both methods t1/2 values for slow repair are approximately constant with radiation dose. At a given radiation dose, the level of initial damage is two- to threefold higher as assessed by the agarose method than by the monolayer method in which DNA repair can occur during the preparation of samples. The detection of higher levels of initial damage by the agarose method permits DNA repair to be assayed at doses as low as 8 Gy and enables fast repair processes to be assayed more readily. However, in the monolayer approach, repair occurs under normal growth conditions and is not subjected to the effects of prior manipulations and/or the rates of nutrient diffusion. Thus this approach might be more representative of normal intracellular repair kinetics.

The use of asymmetric-field inversion gel electrophoresis to predict tumor cell radiosensitivity

The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radio sensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique [21), we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60–80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide: Analysis of five squamous cell carcinoma cell lines derived from head and neck cancers and one normal human fibroblast line showed a significant correlation ( r 2 = 0.92, p ⩽ 0.006) between their extent of DSBR after one hour of repair following 10 Gy of ionizing radiation and their radiosensitivity ( D 0, SF 2Gy). This correlation between DSBR kinetics and radiosensitivity was independent of dose, and there was no correlation between radiosensitivity and damage induction. Thus, AFIGE may overcome some of the problems of clonogenic assays in determining tumor radio sensitivity, particularly if combined with a technique to determine the oxygen profile of individual tumors.

Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced G2 delay

Induction and repair of DNA double-strand breaks (dsb) was investigated in plateau phase Chinese hamster V79 cells and three radiosensitive mutant cell lines derived from them, irs-1, irs-2 and irs-3, using a pulsed-field gel electrophoresis assay, Asymmetric Field Inversion Gel Electrophoresis (AFIGE). There was no difference in the induction of DNA dsb per Gy and dalton between the radiosensitive mutant cells and wild-type V79 cells despite the wide differences in their radiosensitivity. Also, repair of DNA dsb proceeded in all cell lines with similar kinetics. In contrast to these observations at the DNA level, irradiation of exponentially growing cells showed a prolonged delay in G2 for irs-2 cells and a shortened delay in G2 for irs-1 cells, as compared to wild-type V79 cells. These results confirm previous observations suggesting that a deficiency in the rejoining of DNA dsb is unlikely to be the cause of the increased radiosensitivity of irs cells, and implicate alterations in postirradiation cell cycle progression as a possible cause for this phenomenon, although the mechanism is not known.

Level of DNA double-strand break rejoining in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity.

Rejoining of DNA double-strand breaks (dsb) was measured in a dsb repair-deficient mutant of CHO cells, xrs-5, after exposure to various doses of X-rays in the range between 15 and 50 Gy. For the experiments plateau-phase cultures were employed and dsb assayed by a pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE). The half-times of dsb rejoining were larger in xrs-5 than in parental CHO cells and increased in both cell lines with increasing dose of radiation. The fraction of dsb remaining unrejoined after 240 min incubation at 37 degrees C was also higher in xrs-5 than in CHO cells, but decreased with decreasing dose of radiation. Although a decrease in the fraction of unrepaired dsb with decreasing dose has also been reported for repair-proficient cell lines, the extent of the phenomenon and its dependence on dose are entirely different in xrs-5 cells. We propose that this decrease in the fraction of unrejoined dsb with decreasing dose of radiation derives from the genetic alterations underlying the increased sensitivity to radiation of xrs-5 cells, and should be considered whenever results at the DNA level are correlated to results at the cell level. It is likely that similar responses will also be observed in other radiation-sensitive mutant cell lines deficient in dsb repair. There was no difference in the induction of dsb per Gy and dalton, as measured with AFIGE, between CHO and xrs-5 cells tested either in the exponential or in the plateau phase of growth.

Detection of DNA double-strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis.

A pulsed field gel electrophoresis technique, asymmetric field inversion gel electrophoresis (AFIGE), was used to evaluate induction by X-rays of DNA damage in CHO cells. The fraction of DNA activity released from the plug (FAR) was used as a measure for the amount of radiation-induced DNA damage, predominantly DNA double-strand breaks (dsb) (Stamato and Denko 1990), and was determined at various stages of growth and phases of the cell cycle in a range of doses between zero and 70 Gy. The FAR per unit dose fluctuated throughout the cell cycle and was high for cells irradiated in G1; it decreased as cells entered S and reached a minimum in the middle of this phase. The FAR per unit dose increased again as cells progressed towards the end of S, and reached values in G2 similar to those measured in G1. When damage was introduced into DNA by means of 125I decay similar fluctuations in the FAR per decay were observed throughout the cell cycle, suggesting that the variations in the FAR per unit of radiation dose observed throughout the cell cycle do not derive from alterations in the induction of dsb. The fluctuations in the FAR per unit dose throughout the cell cycle were quantitatively similar to the fluctuations in the fraction of activity eluted in irradiated cells assayed by the non-unwinding filter elution assay throughout the cycle (Okayasu et al. 1988), and suggest that both techniques respond to similar DNA replication-associated alterations of the biophysical and/or biochemical properties of the DNA molecule. It is concluded that caution needs to be exercised before differences observed in the FAR between different cell lines or between various phases of the cell cycle after exposure to a given dose of radiation are interpreted as suggesting differences in the induction of DNA dsb.

Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay.

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125I decay accumulation under conditions preventing repair, and is based on the observation that each 125I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5'-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at -80 degrees C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G1, G1/S, mid-S, and G2 + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of 125I decays accumulated during the elapsed period of time. Fluctuations in the FAR per 125I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAR per 125I decay with the FAR per Gy gave an induction of 21 +/- 3, 31 +/- 3, 21 +/- 3 and 26 +/- 8 dsb per Gy per diploid DNA complement for G1, G1/S, S, and G2 + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. 125I decays accumulated at 4 degrees C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by 125I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.

Kinetics of DNA Double-strand Break Repair Throughout the Cell Cycle as Assayed by Pulsed Field Gel Electrophoresis in CHO Cells

Repair of DNA double-strand breaks (dsb) was measured in exponentially growing, plateau-phase and synchronized G1, G1/S, early S, mid-S, late S, G2 + M and mitotic CHO cells. Cells were exposed to 50 Gy X-rays and allowed to repair (up to 4 h) in suspension at 37 degrees C. The dsb rejoining was measured by means of asymmetric field inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis technique. The fraction of DNA-associated [14C]thymidine activity released from the plug (FAR) during electrophoresis was used as a parameter to determine the number of dsb present in the DNA. The assay had been previously calibrated using 125I-decay and its sensitivity in detecting dsb throughout the cell cycle established. Biphasic kinetics of dsb rejoining with a fast and a slow component were obtained throughout the cell cycle, including mitosis, as well as in the various stages of growth. Repair kinetics were described mathematically as the sum of two exponential functions. The repair half-times calculated for the fast component by least-square fitting to the data were in the range of 7-14 min, with the shorter values reached in mid-S and late S and the longer ones during G1 and mitosis. The repair half-times of the slow component were in the range between 60 and 90 min and did not show consistent fluctuations throughout the cell cycle. The fraction of dsb repaired by the slow component ranged from 18% to 37% and did not show consistent variation throughout the cell cycle either. These results suggest that the state of chromatin condensation has only a limited impact on the ability of the cells to rejoin dsb, and indicate that the cell cycle-dependent fluctuations in radiosensitivity cannot be explained by alterations in the rate of rejoining of dsb. The repair half-times of the slow component of dsb rejoining were similar to the half-times of rejoining of chromosome breaks as visualized by the technique of premature chromosome condensation, suggesting a cause-effect relationship between rejoining of this subject of dsb and rejoining of chromosome breaks.

Comparison of DNA Double-strand Break Rejoining as Measured by Pulsed Field Gel Electrophoresis, Neutral Sucrose Gradient Centrifugation and Non-unwinding Filter Elution in Irradiated Plateau-phase CHO Cells

The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.

Asymmetric Field Inversion Gel Electrophoresis: A New Method for Detecting DNA Double-Strand Breaks in Mammalian Cells

A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.

An asymmetric field inversion gel electrophoresis method for the separation of large DNA molecules

An electrophoretic method for separating large DNA molecules which uses periodically inverted electric fields of different magnitude in the two directions is described. Net DNA migration is either in the high field direction or in the low field direction, depending on the relative duration of the pulses. With this approach, molecules of up to 1.6 million base pairs can be separated in parallel lanes after a single run under fixed timing conditions. An inexpensive switching unit is the only device needed in addition to the conventional gel box.