Immobilization of DNA on microporous membranes using UV-irradiation]

Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.     

Reusing nylon membranes for radioactive hybridizations

We describe a procedure for recycling nylon hybridization membranes, enabling their repeated use for radioactive Southern hybridization analysis of different DNA samples. Following hybridization and probe removal, nylon membranes containing covalently linked DNAs were treated with 0.55% sodium hypochlorite. This destroyed the DNA, thereby preventing it from participating in further hybridization and enabling the membranes to be used subsequently for binding new DNA samples. With this procedure, we were able to reuse a single membrane as many as 13 times, with no detectable loss in signal. This method was shown to be effective for membranes supplied by three different manufacturers.     

One-hour downward alkaline capillary transfer for blotting of DNA and RNA.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.     

Optimization of DNA blot hybridization conditions for various types of membranes]

A set of experiments has been conducted to choose the optimal conditions for DNA transfer and fixation on two types of the nitrocellulose, three types of nylon membranes and on capron filters. The buffer capillary transfer systems, electroblotting and gel hybridization are analyzed. Two techniques for DNA binding have been tested under different transfer conditions for all the membrane types: a vacuum fixation at 80 degrees C and a UV-exposure. The results indicate the critical dependence of the efficiency of blot-hybridization on the conditions of UV-treatment. The UV-exposure longer or shorter than the optimal one resulted in a loss of the hybridization efficiency. The optimal DNA-transfer and fixation conditions are recommended for all the membranes tested. The dependence of the optimal transfer and binding conditions on the specific characteristics of different membrane types was demonstrated for maximal sensitivity of the blot-hybridization.     

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.     

Southern transfer of native DNA using nylon membranes

Transfer of native or denatured DNA from gels or filter manifolds was compared using nylon or nitrocellulose membranes. The results were comparable when denatured DNA was used, but only nylon membranes were able to retain native DNA. Although retention of the native DNA was less efficient the bound DNA could be rapidly denatured in situ, avoiding the need to soak gels in alkaline denaturation solution and neutralizing buffer.     

Nick translation of DNA immobilized on nylon membranes

Nick translation of DNA bound to nylon membranes is described. Phage lambda DNA was digested with restriction endonuclease HindIII. The fragments were separated by agarose electrophoresis and electrophoretically transferred to Zeta-Probe nylon membranes. After being air-dried, the areas with DNA fragments attached were cut out and subjected to nick translation. The labeled fragments, removed from the membranes by a single wash step, can be used as specific hybridization probes. Currently used methods require time-consuming electroelution and often additional purification procedures if a specific DNA fragment, separated by gel electrophoresis, is to be labeled by nick translation. With the procedure described it is possible to label many DNA fragments in parallel in a time- and cost-saving manner.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.     

Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system)

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.     

Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization with a cloned viral DNA fragment. The size of the viral DNA was estimated at ca. 158 kilobase pairs. Restriction endonuclease patterns suggested structural similarities to cottontail herpesvirus DNA.     

Relevance of empirical optimization for cross-linking DNA by using an ordinary ultraviolet-light source

For cross-linking DNA onto nitrocellulose or nylon membranes by using ordinary ultraviolet (UV) light, empirical optimization with respect to exposure time is crucial for obtaining complete hybridization signals. Longer than optimal UV exposure reduces the signal intensity particularly in the larger DNA fragments, whereas shorter exposure due to insufficient immobilization produces incomplete signals. Relevance of using ordinary source of UV light with respect to fixing DNA on a large number of circular filters during library screening is discussed.     

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution.

A method is described for the preparation isolation of highly purified adenovirus RNA species. Cytoplasmic RNAs from cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-70 degrees) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This temperature has been found to correlate with the base composition of the DNA fragment. After washing at this predetermined temperature, the specific RNA was eluted at 85 degrees. The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs.     

Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5' end.

Carbodiimide-mediated condensation of DNA onto microwells is investigated. DNA is bound onto the microwells by formation of a phosphoramidate bond between the 5' terminal phosphate group and the microwells. Immobilization of 25 to 30 ng DNA per well is obtained. DNA molecules bound covalently at only the 5' end are, ideally, perfect for hybridization. The practicability of DNA molecules bound to microwells for hybridization is investigated.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Detection of total DNA with single-stranded DNA binding protein conjugates

We have developed a rapid and sensitive method for total DNA measurement using single-stranded DNA binding protein from E coli conjugated with horseradish peroxidase or urease. To detect DNA, the sample is heated or alkali treated to denature the DNA and then filtered through nylon or nitrocellulose membranes. After the single-stranded DNA is bound to the membrane, single-stranded DNA binding protein enzyme-conjugate is incubated with the membrane. Next, the unbound conjugate is washed off the membrane and the bound conjugate detected colorimetrically. The assay can detect 10 pg of DNA in less than 3 hr. This method can be applied to the detection of DNA contamination in therapeutic proteins produced by recombinant DNA or hybridoma techniques.     

Method of extracting DNA from fine needle aspirates of human solid tumors for Southern blot analysis

A rapid, simple, convenient method for extracting DNA from fine needle aspiration (FNA) samples of human solid tumors for Southern blot hybridization studies is described. After the preparation of an air-dried cytologic smear, the remaining sample in the needle was rinsed directly into a test tube for DNA extraction. The extraction procedure, in which manipulation of the sample is minimized, produced sufficient DNA for Southern blot analysis within 24 hours of the FNA biopsy in the ten consecutive cases studied. The DNA bound to the nylon membranes can be washed and reexamined with a variety of probes, allowing studies of lymphoid cell lineage, oncogene amplification or tumor progression. The assessment of cellularity on the cytologic specimen at the time of FNA provided a reliable guide to the need for further passes to obtain sufficient cells for DNA hybridization; the cytologic diagnosis could also be made on the smears.     

Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.     

Quantitative DNA slot blot analysis: inhibition of DNA binding to membranes by magnesium ions.

Titers of wild-type and recombinant adeno-associated viruses are routinely determined by DNA slot blot analysis. The binding of viral DNA to nylon membranes was found to be inhibited by magnesium ions, which are critical components of the DNase I digestion carried out prior to slot blot analysis. Mg2+ions also interfered with the adsorption of plasmid DNA to nylon and nitrocellulose membranes. These observations yield practical insights into the poorly understood mechanisms by which DNA molecules are retained on solid supports.