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.     

Parameters affecting hybridization of nucleic acids blotted onto nylon or nitrocellulose membranes

Nine different nylon and nitrocellulose membranes were compared utilizing four different methods of attaching the nucleic acid target. Nylon membranes repeatedly demonstrated increased sensitivity as compared to nitrocellulose membranes. Sensitivity could also be enhanced by mildly denaturing the target prior to attachment onto the membrane. This was achieved by either UV cross-linking or baking.     

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.     

Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.     

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.     

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.     

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.     

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.     

Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter

Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

Rapid alkaline blot-transfer of viral dsRNAs

The double-stranded genomic RNAs of reovirus and bluetongue virus can be transferred very efficiently from either sodium dodecyl sulfate-polyacrylamide gels or NuSieve agarose gels onto several nylon membranes. After a brief acid depurination treatment, viral dsRNAs from the gels are transferred at room temperature using 0.2 N NaOH as the transfer medium. Four blots can be obtained within 1 h and each blot contains 15-20% of the input RNA sample. These blots can be used immediately without baking in vacuo. Less than 5% of the "fixed" dsRNAs are removed after repeated washings of the membrane blots. As little as 10 pg of the genomic dsRNA segment can be detected in this alkaline Northern blot. A 20- to 50-fold increase in resolution and sensitivity over traditional Northern blots is routinely achieved. These alkaline blots can be reused 6-10 times after appropriate strip washing and proper handling.     

Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.     

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.     

Development of a Membrane-Bound Random DNA Sequence Combinatorial Array Recognition Surface (CARS)

A partially overlapping population of random sequence 60mer DNA molecules consisting of many concatamers of varied lengths was spatially separated in one and two dimensions by electrophoresis in polyacrylamide and transferred to nitrocellulose membranes. The spatially separated library serves as a potential sensor interface on which many different molecular recognition events or target analyte-binding patterns may emerge, thereby theoretically representing a “universal sensor” surface. The separated DNA library has been referred to as a DNA combinatorial array recognition surface or “CARS.” After UV baking and various fluorescence staining or fluorescent probe interactions, the one-dimensional (1-D) and 2-D membrane-bound CARS were digitally photographed and subjected to image analysis with National Institutes of Health Image-Java software. Image analysis demonstrated relatively consistent and more similar spatial fluorescence patterns within CARS analyte treatment groups but noteworthy pattern differences before and after analyte addition and between different analyte treatments. Taken together, these data suggest a potential role for CARS as a novel, inexpensive, self-assembling universal molecular recognition surface that could be coupled to sophisticated Bayesian or other pattern recognition algorithms to classify analytes or make specific identifications, much like the senses of smell or taste.     

Improved northern blot method for enhanced detection of small RNA

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.     

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.     

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.     

Analysis of multiple forms of nuclear factor I in human and murine cell lines.

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.