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.     

Deoxyribonucleases in phytohemagglutinin-stimulated lymphocytes

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by microdisc electrophoresis was used to determine the deoxyribonuclease pattern of human lymphocytes during stimulation with phytohemagglutinin (PHA). Two additional neutral deoxyribonuclease activities are detectable in stimulated cells, one only active with denatured DNA, the other active with native and denatured DNA as substrate, showing a maximum activity after 36 h and increasing in waves respectively. A group of acid deoxyribonuclease activities also shows a maximum after 36 h of stimulation. A neutral deoxyribonuclease active only with native DNA is missing in stimulated lymphocytes. It is suggested that the acid deoxyribonuclease activities and the neutral deoxyribonuclease active only with denatured DNA are involved in DNA synthesis, whereas the involvement of the neutral deoxyribonuclease active with native and denatured DNA in processing of DNA excreted in stimulated lymphocytes is discussed.     

Endonucleases for UV-irradiated and depurinated DNA in barley chloroplasts.

Lysates of barley chloroplasts release more radioactivity into acid soluble form from UV-irradiated and alkylated-depurinated E. coli [3H] DNA than from intact DNA. By means of affinity chromatography on depurinated DNA-cellulose and/or UV irradiated DNA-cellulose and by electrophoresis in polyacrylamide gels, four activities on depurinated DNA were separated. One of these contained activity against heavily UV-irradiated /270 J.m-2/ native DNA. In addition, two other nucleases specific towards UV-DNA were separated. One of them was active on native and heat denatured DNA irradiated with 10 J . m-2 UV, whereas the other was predominantly active on native UV-irradiated DNA.     

Rapid alkaline transfer of low molecular weight DNA from NuSieve GTG agarose gels

The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.     

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.     

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.     

DNA-binding proteins of the malaria vector Anopheles stephensi: purification and characterization of an endonuclease

DNA-binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA-specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg(2+) at pH 7.4. Enzyme activity was completely inhibited by EDTA.     

A comparison of UV cross-linking and vacuum baking for nucleic acid immobilization and retention

The effectiveness of UV cross-linking and in vacuo baking for the immobilization and retention of DNA to various solid supports was investigated. Optimal immobilization treatments for supported and unsupported nitrocellulose and nylon membranes were: UV cross-linking at 254 nm with an exposure of 120 milliJoules/cm2, or baking in vacuo for two hours at 80 degrees C. UV-immobilized nitrocellulose-based membranes showed no increase in sensitivity when compared to baked membranes. An increase in sensitivity was observed for UV-immobilized nylon membranes as compared with baked nylon membranes in some instances, although this varied within lots of the membranes tested. Repeated strippings and heterologous reprobings resulted in loss of target DNA from UV-immobilized nylon membranes as compared to baked nylon membranes. Loss of target DNA from UV-immobilized nitrocellulose-based membranes due to repeated strippings and reprobings was even more pronounced. In vacuo baking of supported and unsupported nitrocellulose and nylon membranes was more effective for immobilization, and more importantly, for retention of target DNA through many reprobings of the same blot.     

Native genomic blotting: a novel approach to mapping DNase I hypersensitive sites and protein-DNA interactions at high resolution

We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.     

Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions.     

Analysis of heat-denatured DNA using native agarose gel electrophoresis.

The use of native or neutral gels to resolve denatured DNA affords a rapid and convenient analytical method for assessing the consequences of a number of procedures employed in molecular biology research. We demonstrate that this method can be used to analyze transition melting temperature (Tm) and strand breakage in heat-denatured duplex DNA. This shows that some commonly recommended denaturation procedures can result in significant degradation of DNA and that reannealing or aggregation can occur when samples are concentrated or ionic conditions altered.     

Semi-dry electroblotting of DNA

A procedure is described for the rapid transfer of DNA from agarose gels to nylon membranes using the semi-dry electroblotting technique. A Hind III digest of lambda DNA which was separated in a 1% agarose gel containing Tris, Borate, and EDTA (pH 8.0) was employed for the electrotransfer experiments. Transfer efficiency was determined by staining the DNA on the nylon membranes with a colloidal iron reagent. Current densities of 3-5 mA/sq. cm of gel permitted the transfer of high (23 kb) and low (0.3 kb) molecular weight fragments within 15 min. However, efficient transfer required a high ionic strength buffer that would prevent uneven dehydration of the agarose gel. Critical parameters for the transfer of nucleic acids with the semi-dry technique are discussed.     

"Pocket blotting": a method for transferring nucleic acids onto nylon membranes

We have developed a fast and efficient method for transferring nucleic acids onto nylon membranes. This method requires less DNA for transfer; no decrease in efficiency is observed after successive probing, and several gels can be processed simultaneously. We believe that this techniques is of general interest in routine analysis of multiple samples in population genetic studies or in diagnosis purposes.     

Quantitative molecular hybridization on nylon membranes

A study of DNA hybridization to DNA covalently bound to nylon membranes was made in order to develop a quantitative method for molecular hybridization using a nylon-based matrix. Chloroplast DNA was covalently attached to nylon membranes by irradiation at 254 nm. Under hybridization conditions the initial rate of DNA loss from the nylon membranes was 5-10% per 24 h, while under comparable conditions DNA bound to nitrocellulose membranes was lost at a rate of 38 to 61% per 24 h. Several sets of hybridization conditions were examined to select one giving reasonable hybridization rates and minimal loss of bound DNA. Under the conditions selected [Denhardt's solution (D. Denhardt, 1966, Biochem. Biophys. Res. Commun. 23, 641-646), 0.5 M NaCl, 0.1% sodium dodecyl sulfate, and 31.4% formamide at 50 degrees C for 92 h], hybridization was observed to be 29% more efficient on nylon membranes than on nitrocellulose. Several attempts to remove previously hybridized DNA from nylon membranes proved only partially successful. Reuse of the membranes, therefore, was of limited value. Quantitative hybridization of total radiolabeled tobacco cellular DNA to cloned tobacco chloroplast DNA attached to nylon yielded results similar to those previously reported using nitrocellulose membranes. However, use of nylon membranes greatly facilitated the manipulations required in the procedure.     

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.     

Separation of the complementary strands of DNA fragments on polyacrylamide gels.

32P-labeled (in vivo) phiX174 RFI DNA was restricted by Hinc II. Three aliquots of the same digest: a) nondenatured, b) heat denatured, and c) denatured by 5 mM Me-HgOH were analyzed on 3-15% acrylamide gel gradients or on 3% gels with reduced N,N'-methylene-bis-acrylamide. The autoradiography of the gels showed that the nondenatured sample migrates two times faster than the denatured samples. After denaturation each original fragment appeared as a doublet. Using in vitro synthesized RFI DNA labeled only in negative strand with 32P we could identify the position of the negative strand in each denatured doublet. The single strand DNA fragments could be recovered from the gel slices on a semi-preparative scale by electrophoresis into dialysis tubing.     

Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis.

A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

BKm minisatellite sequences are not sex associated but reveal DNA fingerprint polymorphisms in rainbow trout

GATA-GACA repetitive sequences first isolated from a female snake (termed BKm sequences) and associated with sex chromosomes in some species were hybridized to DNA from rainbow trout (Salmo gairdneri). Genomic DNA was studied from three groups of rainbow trout: (i) randomly selected males and females from an outbred group, (ii) androgenetic individuals from an inbred strain, and (iii) parents and offspring of an outbred strain. Three restriction enzymes (EcoRI, HaeIII, or HinfI) were used to digest the genomic DNA. The DNA was electrophoresed in agarose gels, transferred to nylon membranes, and the GATA-GACA repetitive sequence probe was hybridized to this DNA. There was no evidence of sex-associated patterns of hybridization with the enzymes used. However, the sequences reveal DNA fingerprint polymorphisms which appear to be inherited in a stable manner.     

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.