ISSN:0173-0835    

DOI:10.1002/elps.1150100502

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal‐field‐alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120 s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presen

ISSN:0173-0835    

DOI:10.1002/elps.1150100503

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe electrophoretic separation of DNA molecules can be controlled by the use of contour‐clamped homogeneous electric fields (CHEF). This paper describes an improved CHEF apparatus with negligible distortion in the electric fields. When the electric field was periodically reoriented, DNA molecules up to 2 megabases were resolved in a highly uniform manner. Furthermore, when the field strength was changed with orientation, topological variations of conventional‐sized DNA molecules were resol

ISSN:0173-0835    

DOI:10.1002/elps.1150100504

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes fromS. pombeandS. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.

ISSN:0173-0835    

DOI:10.1002/elps.1150100505

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters.

ISSN:0173-0835    

DOI:10.1002/elps.1150100506

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe mobility of linear DNA during field‐inversion gel electrophoresis was measured as a function of molecular weightMr, pulse timet, and field strengthE.Values ofMrbetween 48.5 and 194 kilobase pairs (kb),Efrom 5 to 14 V/cm and pulse times of 0.3 to 12 s were used. The data are presented as three‐dimensional surfaces of mobility:E:tfor fixedMror graphs of mobility:Mr:tfor fixedE.The surfaces are not smoothly increasing functions ofE,Mr, ortbut instead show a valley with minimum mobility and a steep rise in mobility astincreases. For a field of 10 V/cm, 1 % agarose gels, and 3:1 ratio of forward: back pulse time, the forward switching timet* at which the mobility changes most rapidly is given byt* =(0.034 ± 0.003)MrforMrin kb andt* in seconds. The data and equations delineate the best conditions to achieve a particular separa

ISSN:0173-0835    

DOI:10.1002/elps.1150100507

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe maximum length of DNA molecules that can be separated by gel electrophoresis can be increased greatly by periodically altering the direction of the electric field with respect to the gel by an angle that exceeds 90°. One method involves rotating the gel by the desired angle in alternate directions periodically during electrophoresis. We describe a modification of the rotating gel electrophoresis apparatus developed by Serwer (Electrophoresis1987, 8, 301–304) that uses a pneumatic rotary actuator instead of a stepping motor, hence reducing the cost by about 50 %. Other advantages of our design are a lower center of gravity that makes the apparatus more stable and the removal of all electrical power from beneath the fluid‐filled electrophoresis chamber. We present data demonstrating the separation of chromosomal length DNA molecules fromSaccharomyces cerevisiaestrain 334 into 14 resolved bands in parallel l

ISSN:0173-0835    

DOI:10.1002/elps.1150100508

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single‐ and double‐strand breaks formed by γ‐rays for DNA irradiatedin vitro. For single‐strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double‐strand break doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single‐strand breaks is linear while the dose response function for double‐strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with s

ISSN:0173-0835    

DOI:10.1002/elps.1150100509

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractAgarose gel electrophoresis is used to fractionate linear, double‐stranded DNA by its length. Sieving of the gel is the cause of this fractionation and has been investigated by developing theoretical models and by quantifying sieving observed during electrophoresis. Here are reviewed the following aspects of the fractionation of linear, double‐stranded DNA by agarose gel electrophoresis: (1) the basic observations that qualitatively characterize these fractionations, (2) evidence for the deformation of DNA's random coil, (3) quantitative analysis of the relationship of observed electro‐phoretic mobility to the DNA's length, (4) theoretical models that have been developed to explain data presented in Sections 1–3, (5) observations not yet quantitatively explained by models, and (6) some aspects of the use of a variable electrical field (pulsed‐field gel electrophoresis) to improve se

ISSN:0173-0835    

DOI:10.1002/elps.1150100510

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractTransient electric birefringence has been used as an analytical tool to study the orientation of DNA in agarose gels, and to study the orientation of the matrix alone. The sign of the birefringence of DNA oriented in an agarose gel is negative, as observed in free solution, indicating that the DNA molecules orient parallel to the direction of the electric field. If the median pore diameter of the gel is larger than the contour length of the DNA molecule, the DNA effectively does not see the matrix and the birefringence relaxation time is the same as observed in free solution. However, if the median pore diameter of the gel is smaller than the contour length of the DNA, the DNA molecule becomes stretched as well as oriented. For DNA molecules of moderate size (⩽ 4 kb), stretching in the gel causes the birefringence relaxation times to increase to the values expected for fully stretched molecules. Complete stretching is not observed for larger DNA molecules. The orientation and stretching of DNA molecules in the gel matrix indicates that end‐on migration, or reptation, is a likely mechanism for DNA electrophoresis in agarose gels.When the electric field is rapidly reversed in polarity, very little change in the orientation of the DNA is observed if the DNA molecules were completely stretched and had reached their equilibrium orientation before the field was reversed in direction. Hence completely stretched, oriented DNA molecules are able to reverse their direction of migration in the electric field with little or no loss of orientation. However, if the DNA molecules were not completely stretched or if the equilibrium orientation had not been reached, substantial disorientation of the DNA molecules is observed at field reversal. The forced rate of disorientation in the reversing field is faster than the field‐free rate of disorientation. Complicated patterns of reorientation can be observed after field reversal, depending on the degree of orientation in the original field direction.The effect of pulsed electric fields on the orientation of the agarose gel matrix itself was also investigated. If very short pulses of high amplitude (e.g.1–10 kV/cm in amplitude and 10–1000 μs in duration) were applied to the gel, the sign of the birefringence was small and positive, the Kerr law was obeyed, and the birefringence decayed to zero after the removal of the electric field with a relaxation time varying from 10–220 μs, depending on the length of the orienting pulse. These results indicate that individual agarose chains or bundles of chains, or dangling ends of the matrix, could be oriented by very short pulses of high amplitude. However, when smaller electric fields were used (e.g., 10–100 V/cm applied to the gel for 0.5–2 s), the amplitude of the birefringence of the gel matrix increased markedly and the signal passed through an extremum several seconds after removal of the electric field before decaying slowly to zero. These slow time‐dependent effects indicate that domains in the agarose matrix were being oriented by the longer pulses used at the lower electric field strengths. The sign of the birefringence of the agarose gel could be reversed from positive to negative, orvice versa, by reversing the direction of the applied electric field, indicating that the domains apparently change their direction of orientation from parallel to perpendicular (orvice versa) after field reversal. Orientation and reorientation of microdomains of the matrix in alternating pulsed electric fields would increase the fluidity of the matrix, making it easier for very large DNA molecules to migrate through the gel dur

ISSN:0173-0835    

DOI:10.1002/elps.1150100511

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY