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.     

Characterization of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha purified from calf thymus using monoclonal antibody.

Existence of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha was shown by production of a monoclonal anti-calf thymus 10S DNA polymerase alpha antibody secreted from a hybridoma line named 3H1. The antibody bound three polypeptides with Mr = 180,000, 56,000 and 32,000 in hydroxylapatite fraction of 10S DNA polymerase alpha by immunoblot. The antibody co-precipitated the polypeptides with the large polypeptide (Mr = 150,000-140,000) of 10S DNA polymerase alpha with the aid of second antibody. Among three polypeptides, the Mr = 56,000 polypeptide was co-purified with DNA polymerase alpha through DNA-cellulose chromatography and repeated sucrose rate-zonal centrifugations. The Mr = 56,000 polypeptide was still associated with 10S DNA polymerase alpha after second sucrose rate-zonal centrifugation, but the amount of it was reduced. The polypeptide was banded at pH 7.2-8.0 and displayed microheterogeneity in respect of isoelectric point by isoelectrofocusing with 7 M urea, and showed weak DNA-binding property after blotting onto a nitrocellulose. The antibody against the polypeptide precipitated DNA polymerase alpha from human, rat, and mouse, and Mr = 56,000 and 32,000 polypeptides were detected in these DNA polymerase alpha fractions by immunoblot. These results suggest that the polypeptide with Mr = 56,000 may take part in the DNA polymerase reaction.     

Non-radioactive diagnosis of viral infections using "DNA-peroxidase" probes]

DNA-peroxidase probes were synthesized according to a modified method (Renzetal) for the detection of lambda phage DNA (model system), polio, potato X and M, tobacco mosaic viral RNAs by spot hybridization onto nitrocellulose membranes. cDNAs (300-1400 bases) complementary to the viral RNAs were cloned in M13 phage DNA or pTZ19. Efficacy of each step of the probe construction and the diagnostic procedure were thoroughly examined. Peroxidase activity manifested with non-toxic stain (NTS) was 3-5 fold more sensitive in comparison with alpha-Cl-naphthol or bisanisidine. It was found that HRP became much more stable to heat in diluted samples and was 2-3 fold more active after coupling with polyethylene imine spacer. Also, sodium borohydride reduction of the cDNA and PEI-HRP adduct crosslinked by the glutardialdehyde resulted in the stabilization of the probes. Target nucleic acids or diagnostic samples were efficiently fixed onto nitrocellulose membranes by a short-time UV irradiation. Diagnostics of cellular extracts with the preliminary prepared probes takes 4-5 hours due to express hybridization (1 hr) with 100-200 ng/ml of specific nucleotide sequence. Up to 20 pg (less than 10(-17) M) of the purified viral nucleic acids and 30-50 pg of them in the total fraction of the cellular nucleic acids isolated from the infected cells were identified with the probes. 50-10000 fold diluted lysate of the HeLa cells infected with poliovirus (PV1) and both crude extracts of potato tuber or potato and tobacco leaf tissues infected with PVX, PVM or TMV displayed specific signals with the respective DNA-HRP probes.     

Large-scale isolation of plasmid DNA and purification of lambda phage DNA using hydroxylapatite chromatography

A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 M, the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. lambda Phage DNA is also easily eluted from hydroxylapatite under the conditions used.     

Open-pulled straw (OPS) vitrification of mouse hatched blastocysts

This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions - that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)--at 25 degrees C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs.     

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.     

Reorganization of the protein composition of the nuclear matrix of hepatoma cells after inhibition of DNA synthesis with novobiocin

The nuclear matrix of Zajdela hepatoma cells, in which DNA synthesis was blocked by novobiocin, contained 2.5-3.0 times more DNA and protein not dissociating in 2 M NaCl than the nuclear matrix of control cells. Chromatography of nuclear matrix preparations on Sepharose 2B-CL resulted in isolation of tightly bound DNA-protein complexes which did not dissociate in 8 M urea or 0.1% SDS. Subsequent elution of DNA-protein complexes on a hydroxylapatite column with a buffer containing 4 M guanidine hydrochloride and 5 M urea caused partial dissociation of the complexes. Electrophoretic analysis revealed essential changes in the composition of proteins DNA-protein complexes of hepatoma cells nuclear matrix during inhibition of DNA synthesis.     

A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.     

Taste responses of Drosophila melanogaster

The amounts of sugar solution consumed by Drosophila melanogaster flies were determined. Starved and desiccated flies of a wild type strain (QA) consume 7?9 × 10^?2 λ of a 0.3 M sucrose solution per fly during the first hour and less later. They consume more of the 0.3 M sucrose solution than of the more diluted and the more concentrated solutions. In preference-aversion tests the flies discriminated between water and various sugar solutions, and between different sugar concentrations. Contrary to other fly species these flies did not prefer 0.05 M fructose over 0.05 M glucose. 0.3–0.5 M NaCl added to 0.1 M sucrose turned a preference over 0.01 sucrose into an aversion. A mutant, Lot-94, selected for its increased consumption of a 1 M NaCl solution was found to consume more of all test solutions. The amount of NaCl that had to be added to 0.1 M sucrose to turn the preference over 0.01 M sucrose by the mutant flies into aversion was not different from that found for the wild type flies.     

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.     

A refined method for silver staining of nucleic acids fixed on nitrocellulose

A refined silver staining method was developed to stain nucleic acids fixed onto nitrocellulose membranes and nylon-based membranes. Approximately 4 ng RNA or DNA can be stained with this method with no protein interference. This method involves simple repetition of immersions of membranes in three solutions prepared from common chemicals. The total staining time is less than 30 min.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

The affinity adsorptive recovery of an infectious herpes simplex virus vaccine

The chromatographic purification of a recombinant Herpes Simplex Virus (type 2) from salt- and heparin-released harvests of infected complementing Vero (CR2) cells is addressed. Functionalized matrices and process operating conditions are identified that provide adequate virus titres in eluates that are significantly reduced in CR2 cell protein and DNA and possess a low level of HSV-2 protein. Virus from diluted salt-released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin-Sepharose or Cellufine-heparin matrices but was virtually completely adsorbed onto Cellufine-sulfate and heparin-HP matrices. Virus was recovered by either a linear salt gradient elution (0.14-2 M NaCl) or by a single-step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices. Virus from undiluted salt-released harvest (0.8 M NaCl) was substantially adsorbed onto Cellufine-sulfate gel (44% adsorption) and completely adsorbed onto heparin-HP matrices. This virus was recovered with high yield by either gradient or step elution with phosphate-buffered saline. Finally, heparin-harvested virus was fed directly to these matrices and quantitatively adsorbed. The virus could be completely recovered from the heparin-HP matrix with 1.5 M NaCl buffer to provide a purified preparation containing only 0.05 pg protein/pfu and 1.2 x 10(-4) pg DNA/pfu.     

The impact of urea on viscosity of hydroxethyl cellulose and observed mobility of deoxyribonucleic acids

The influence of urea on the viscosity of hydroxyethyl cellulose (HEC), and the state and separation of double-stranded DNA, was studied by viscometry, fluorometry, and capillary electrophoresis. The results show that double logarithm plots of specific viscosity against the volume fraction of HEC in very dilute polymer solutions are linear, the slopes of which decrease from 0.96 in 0M to 0.29 in 7M urea. The linear regression plots converge at 0.0029 g/mL, the entanglement threshold of HEC. The inclusion of urea in HEC solution thus provides an accurate method of determining its entanglement threshold from such plots. Above the entanglement threshold of HEC, urea has no effect on the specific viscosity of HEC. Results also show that urea has no effect on double-stranded DNA. No change in fluorescence was observed when increasing amounts of urea were added to a fixed concentration of DNA. To examine the influence of urea on the migration of DNA in HEC, the separation of DNA was carried out by polymer-solution capillary electrophoresis in HEC solutions containing 0 or 7M urea using unmodified capillary. Observed mobilities were used in data reduction. It was found that a parallel relationship exists between the observed mobilities and the true mobilities. In buffers containing no urea, the pseudo-free solution mobility appears to be independent of the DNA size. It was also observed to be independent of the electric field below 300 V/cm, but relates exponentially to it in 7M urea. The pseudo-retardation constants obtained by Ferguson-like plots were observed to be positive for smaller DNA molecules below 300 V/cm and increasing linearly with electric field in 0M urea, but nearly constant in 7M urea.     

Somatic embryogenesis on Thin Cell Layers of a C4 species,Digitaria sanguinalis (L.) Scop

Somatic embryogenesis was obtained from transverse thin cell layers (tTCLs) of Digitaria sanguinalis. tTCLs (0.2 - 0.4mm thick, 1mm in diameter) were excised from 4-week-old seedlings and placed onto Murashige and Skoog media supplemented with a varying concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (from 1 μM to 100 μM) and sucrose (from 3% to 24%). Somatic embryos were obtained in the dark 7-10 days after inoculation from tTCLs excised at specific levels on the seedling and cultured in the presence of 2,4-D (5 μM to 10 μM) and sucrose (3 to 6%). The exposure of the tTCLs to light decreased the percentage of tTCLs forming somatic embryos. Viable plantlets were obtained 2 weeks after transfer onto a cytokinin-containing medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of formaldehyde on the efficiency of hybridization of DNA immobilized on nitrocellulose filters.

We report in this study that under certain conditions formaldehyde interacts with DNA and makes it more efficient for hybridization on nitrocellulose filters. Hybridization signals of formaldehyde-treated DNA are stronger (up to 10 fold) as compared with that of the heat- or alkali-denatured DNA. Various parameters of the DNA-formaldehyde reaction are optimized as follows: (a) 6 x SSC, 10% formaldehyde, 60 degrees C, 20-30 min, reaction volume 10-200 microliters or (b) 6 x SSC, 5% formaldehyde, 98 degrees C, 15 min, reaction volume 10-200 microliters. Treatment of agarose gels after electrophoresis with formaldehyde improved both the transfer of DNA and the efficiency of hybridization. The following conditions are recommended for gel treatment: denaturation in 0.3 N NaOH, 1 M NaCl followed by neutralization with 0.5 M phosphate buffer, pH 7.0, containing 10% formaldehyde at 60 degrees C for 20 min.     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.