Electroblotting of double-stranded DNA for hybridization experiments: DNA transfer is complete within 10 minutes after pulsed-field gel electrophoresis

A rapid, simple, and efficient method for DNA blotting is presented. This method is characteristic in that DNA is transferred without denaturation from an electrophoretic gel to a membrane in low-salt concentrations and denatured on the membrane after blotting. More than 89% of double-stranded DNAs ranging in size from 75 to 1.9 million bp can simultaneously be transferred from the gel to a positively charged nylon membrane within 10 min. The present "low-salt electroblotting" method is superior to other blotting methods in that it saves time and labor and its high and even transfer efficiency makes it useful for hybridization analysis.     

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.     

Rapid transfer of DNA from agarose gels to nylon membranes.

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.     

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.     

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.     

Dynamic behavior of large DNA molecules in an aqueous solution toward the understanding of pulsed-field electrophoresis

Conformational dynamics of large DNA molecules have been monitored by use of a fluorescence microscopy. The time-trace of the movement has been quantitatively analyzed with Fourier transformation.     

Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes.

A method for transferring small DNA fragments from composite polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM)-paper is described. DNA fragments are separated by electrophoresis in polyacrylamide-agarose gels crosslinked with N,N′-diallyltartardiamide instead of N,N′-methylenebis-acrylamide. The crosslinks are cleaved by treating the gel with periodic acid after electrophoresis and the DNA is denatured with alkali. After neutralization, the single stranded DNA fragments are transferred to DBM-paper and detected by hybridization with labeled DNA probes. The procedure has been used to transfer and visualize unlabeled SV40 DNA fragments in the size range 28 to 1790 base pairs.     

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.     

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.     

Direct hybridization of labeled DNA to DNA in agarose gels

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.     

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.     

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.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis

We have designed a new device [Biotrap (Elutrap in the U.S.A. and Canada), available from Schleicher & Schuell] for electroelution, -concentration , and -dialysis of DNA and other charged macromolecules above 5 kDa. In an electric field, the DNA migrates in an open channel out of the gel slice through a microporous membrane, BT2, into the trap section, where it is retained by a very dense, non-adsorbant, and inert membrane BT1. Specifically designed for use in an electric field, the matrix of this new membrane is much denser than that of dialysis membranes. In contrast to dialysis membranes, BT1 will not adsorb large DNA fragments nor allow passage of small DNA fragments when subjected to an electric field. In the absence of an electric field, BT1 and BT2 effectively seal the trap, maintaining the final elution volume of the purified sample. The trap can contain from 200-600 microliter and is collected from above with a pipet. In the experiments described here, 85-95% of oligonucleotides (14-mer) and large (150 kb) DNA fragments were recovered, independent of fragment length. The purity of the eluted DNA was demonstrated by restriction enzyme digestion, nick-translation, primer extension, end-labeling with polynucleotide kinase, and ligation. Electrodialysis was successfully used for the complete removal of common contaminants inhibiting the polynucleotide kinase reaction and for the removal of CsCl from DNA samples.     

Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells.

We have identified two novel enzyme systems in human HeLa nuclear extracts that can nick at specific sites of DNA molecules with base mismatches, in addition to the T/G mismatch-specific nicking enzyme system (Wiebauer, K., and Jiricny, J. (1989) Nature 339, 234-236). One enzyme (called all-type) can nick all eight base mismatches with different efficiencies. The other (A/G-specific) nicks only DNA containing an A/G mismatch. The all-type enzyme can be separated from the T/G-specific and A/G-specific nicking enzymes by Bio-Rex 70 chromatography. Further purification on a DEAE-5PW column separated the A/G-specific nicking enzyme from the T/G-specific nicking enzyme. Therefore, at least three different enzyme systems are able to cleave mismatched DNA in HeLa nuclear extracts. The all-type and A/G-specific enzymes work at different optimal salt concentrations and cleave at different sites within the mismatched DNA. The all-type enzyme can only cleave at the first phosphodiester bond 5' to the mispaired bases. This enzyme shows nick disparity to only one DNA strand and may be involved in genetic recombination. The A/G-specific enzyme simultaneously makes incisions at the first phosphodiester bond both 5' and 3' to the mispaired adenine but not the guanine base. This enzyme may be involved in an A/G mismatch-specific repair similar to the Escherichia coli mutY (or micA)-dependent pathway.     

A method for the recovery of DNA from agarose gels.

We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.