Computer-controlled discontinuous rotating gel electrophoresis for separation of very large DNA molecules

The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described. The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles. The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field. Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good. Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed. In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges. Field inversion gel electrophoresis is possible as well. The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains.     

A rhelogical separator for very large DNA molecules.

We present a rheological separation method for DNA molecules in which their deformability is used to advantage. This is the "radial migration method"; here we present experimental verification of the principle, theory having been reported elsewhere. The main conclusions are: (1) the theory is reasonably good; (2) radial migration is highly sensitive to the molecular weight, as predicted, and (3) intact T2 DNA (1.25 X 108 daltons) can be made to migrate about three centimeters in less than three hours.     

Ice-water interface migration by temperature controlling for stretching of DNA molecules

This report shows a new DNA stretching method using migration of an ice-water interface. DNA molecules were stretched accompanying the migration of the solid-liquid interface and immobilized in frozen area. This simple method needs no chemical modification to keep DNA in the stretched form. For full stretching of DNA molecules, one terminus of the DNA molecules were anchored on silanized substrate. The anchored DNA molecules were stretched by freezing the DNA solution. The stretched DNA molecules were observed after sublimation of the frozen solution keeping its stretched form on silanized surface which had no attractive interaction with DNA molecules except for the SH-modified terminus in solution. An infrared (IR) laser beam was introduced to a frozen DNA solution through an objective lens for local area melting of the solution. Scanning of the laser irradiation caused stretching and enclosing of DNA molecules in the frozen area followed by migration of the solid-liquid interface.     

DNA binding fluorescent proteins for the direct visualization of large DNA molecules

Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (K_d = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells.     

A DNA prism for high-speed continuous fractionation of large DNA molecules

The analysis and fractionation of large DNA molecules plays a key role in many genome projects. The standard method, pulsed-field gel electrophoresis (PFGE), is slow, with running times ranging from 10 hours to more than 200 hours. In this report, we describe a thumbnail-sized device that sorts large DNA fragments (61-209 kilobases (kb)) in 15 seconds, with a resolution of approximately 13%. An array of micron-scale posts serves as the sieving matrix, and integrated microfluidic channels spatially shape the electric fields over the matrix. Asymmetric pulsed fields are applied for continuous-flow operation, which sorts DNA molecules in different directions according to their molecular masses, much as a prism deflects light of different wavelengths at different angles. We demonstrate the robustness of the device by using it to separate large DNA inserts prepared from bacterial artificial chromosomes, a widely used DNA source for most genomics projects.     

Radial migration of DNA molecules in cylindrical flow. I. Theory of the free-draining model

Inward radial migration of large DNA molecules undergoing concentric-cylinder shear flow has been previously observed in this laboratory during measurements of viscoelastic retardation times. In this paper a theoretical treatment of this effect is presented in the framework of the bead-spring model of polymer molecules. Equations of motion of the diffusion type are solved for the free-draining polymer in a solvent undergoing Couette flow. The results show an inward radial motion, essentially due to the curvilinear solvent flow field.     

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR^– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.     

Transformation of Escherichia coli with large DNA molecules by electroporation.

We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e. molecules > 100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA. Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested.     

Slalom chromatography: size-dependent separation of DNA molecules by a hydrodynamic phenomenon

Slalom chromatography, a size-dependent DNA fractionation method based on a new principle [Hirabayashi, J., & Kasai, K. (1989) Anal. Biochem. 178, 336-341], was systematically studied in detail. In this method, larger DNA fragments are eluted much later than smaller ones from columns packed with spherical microbeads. Elution of a series of DNA fragments was systematically examined by using columns packed with polymer-based packings of different diameter and different pore size for high-performance gel permeation chromatography. Packings of smaller diameter proved to be superior for resolving the smaller size range of DNA, while the reverse was the case for larger DNAs. Application of a faster flow rate led to larger retardation of every DNA fragment, while at the lowest flow rate applied (0.067 cm/min), all the fragments were eluted almost at the void volume. When the column temperature was lowered, retardation of DNA became larger. On the other hand, differences in the chemical nature and the pore size of packings, or in the hydrophobicity of the eluting solvent, had little effect on DNA retardation. Size-dependent fractionation of DNA was also achieved even on columns packed with nonporous packings having anionic groups (cation exchangers). In conclusion, these results confirmed the previous conclusion that slalom chromatography is not based on an adsorption or equilibrium phenomenon but should be attributed to a hydrodynamic phenomenon.     

Separation of large DNA molecules by modified pulsed field gradient gel electrophoresis

Resolution of DNA fragments by pulsed field gradient gel electrophoresis is a function of the pulse time, geometry, and strength of the orthogonal electric fields. The first field geometry described had a number of disadvantages. We show that these disadvantages can be largely overcome by a modified electric field geometry together with an altered switch pattern. These changes are shown to have critical consequences for the technique. Resolution is more uniform across the gel, which permits more samples to be analyzed on the same gel. In addition, DNA molecules follow a migration path that is approximately straight down the gel. This aspect also increases the number of usable wells. One important property of the system described here provides some insight into the mechanism whereby DNA molecules are resolved by this method.     

Preparation of multimilligram quantities of large, linear DNA molecules for structural studies

We describe a method for preparing large, linear DNA molecules in amounts that are suitable for structural studies. The procedure employs self-primed DNA amplification on a starting molecule that consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of the DNA molecule of interest. Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interest with minimal restriction on length and sequence.     

The termination region for SV40 DNA replication directs the mode of separation for the two sibling molecules

Separation of the two newly replicated chromosomes in simian virus 40 late replicating intermediates (RI*) occurred by two pathways. The parental DNA strands were completely unwound, releasing circular DNA monomers with a gap in the nascent strand (Form II*), or duplex DNA in the termination region was not unwound, resulting in formation of catenated dimers. Under optimal conditions, both products were transient intermediates in replication, although Form II* was predominant. However, in hypertonic medium both RI* and catenated dimers accumulated, and Form II* was not observed. Hypertonic medium appeared to inhibit both DNA unwinding in the termination region and separation of catenated dimers. When the size of the genome or the position of the origin of replication was changed, termination occurred at sites other than that of wild-type SV40. Neither catenated dimers nor RI* DNA accumulated at these sites. Instead, RI* separated into Form II*. Unwinding parental DNA was more difficult at some termination regions than others. Therefore, although completion of DNA replication does not require a unique termination sequence, this sequence can determine the mode of separation for sibling molecules.     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers, and (4) stop signals locally expressed in the outmost layer of cortex.     

Rapid pulsed field separation of DNA molecules up to 250 kb.

Pulsed field gel electrophoresis (PFGE) is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kilobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90 degrees to be effective. However, reorientation angles of 90 degrees and even less will resolve DNA fragments a few hundred kb and smaller approximately 5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90 degrees reorientation angles is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120 degrees. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large insert bacterial clones, such as those using cosmid, Fosmid, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.     

An explanation for rotor speed effects observed during sedimentation of large folded DNA molecules

A model is proposed to account for the changes in sedimentation coefficient (s) that are observed when isolated bacterial micleoids are sedimented at different rotor speeds. It is suggested that the terminal of DNA loops in the chromosome become analogous hydrodynamically to the ends of a linear DNA double-helix. The loops then become more extended at higher rotor speeds resulting in an increased fractional coefficient. Application of the Zimm (1974) equation to this model shows that the predicted changes in s values are consistent with those observed. Similar changes in s values occur when the chromosome is sedimented in rotors of different geometry including a zonal rotor. Thus, the changes are not due to wall interactions.