A discontinuous buffer system increasing resolution and reproducibility in DNA sequencing on high voltage horizontal ultrathin-layer electrophoresis

A novel discontinuous buffer system for DNA sequencing based on horizontal ultrathin-layer gel electrophoresis is described. The optimized system, named unbuffered stacking gel/discontinuous borate EDTA-buffer system, is composed of a 0.5 mm thick stacking gel, where standard sequencing reactions (1 microL volume) are easily loaded, and a 50 microns ultrathin running gel, where DNA fragments are separated. The novel discontinuous buffer system allows for sample concentration and efficient injection from the stacking gel into the capillary slab gel. Increased resolution, assessed by autoradiography, can be achieved within 25 min running time already over a 10.1 cm distance from the gel slot compared to the conventional gel system. An advantage of the new system is the capacity to resolve compressions in GC-rich regions, usually causing migrating artifacts in standard gels. The described system affords a major improvement in speed, resolution and reproducibility in DNA sequencing.     

Disc-crossed immunoelectrophoresis. A simple "laying-on" technique permitting the use of commercially available agarose

A technique, disc-crossed immunoelectrophoresis, is described to provide a simple and convenient means of performing electrophoresis in polyacrylamide gel in a discontinuous buffer system (disc electrophoresis) followed by electrophoresis into antibody-containing agarose gel. A simple washing of the polyacrylamide gel in distilled water combined with a "laying-on" method, seems to be a satisfactory and rapid way to obtain immunoprecipitation patterns of high quality using commercially available agarose in the second electrophoresis.     

A simplified polyacrylamide gel electrophoresis apparatus for simultaneous application of multiple buffer systems or detergent combinations

A previous design of an apparatus for the simultaneous fractionation by polyacrylamide gel electrophoresis in 10 different buffer systems (1) was replaced by a greatly simplified new design, employing small, cylindrical buffer partitions within the lower buffer reservoir and/or upper buffer reservoir of a conventional, temperature-regulated polyacrylamide gel electrophoresis apparatus for cylindrical gels. The apparatus was tested in application to the problem of simultaneous polyacrylamide gel electrophoresis in different buffer systems with the purpose of optimizing the operative pH for a particular fractionation problem. It was also applied to fractionations in a single buffer system to which various combinations of ionic and nonionic detergents were admixed.     

Formyl-methyl-methylenetetrahydrofolate synthetase-(combined). An ovine protein with multiple catalytic activities

Formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, and methylenetetrahydrofolate dehydrogenase in sheep liver co-purify 200-fold to yield a homogeneous preparation containing a single protein species observed on discontinuous polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The synthetase and dehydrogenase activities migrate with the single protein band observed on polyacrylamide gel electrophoresis in two discontinuous buffer systems near pH 8. The protein sediments with a single symmetrical boundary with sedimentation coefficient (S20,w) = 8.30 S and elutes from molecular exclusion chromatography columns at a position corresponding to a diffusion coefficient (D20,w) = 3.99 X 10(-7) cm2 sec-1. Dodecyl sulfate-polyacrylamide gel electrophoresis of the protein intramolecularly cross-linked with dimethylsuberimidate shows one protein species of Mr = 218,000 in addition to the species of Mr = 108,500 characteristic of the unmodified protein. NH2-terminal analysis of the protein using 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chloride in the presence of dodecyl sulfate results in the recovery of a single dansyl amino acid, alanine. The three activities are thus shown to reside in a protein composed of two apparently identical subunits, and the trivial name formyl-methenyl-methylenetetrahydrofolate synthetase(combined) is proposed for this enzyme to suggest the multiple catalytic activities associated with the single protein species.     

SDS agarose gels for analysis of proteins

A new agarose-based protein electrophoresis gel system is described. The system consists of a highly resolving agarose, MetaPhor XR (FMC BioProducts, Rockland, ME, USA) dissolved in urea and TBE buffer and a stacking gel composed of a high gel-strength agarose, SeaKem Gold (FMC BioProducts). TBE containing sodium dodecyl sulfate (SDS) is used as electrophoresis buffer. The disadvantages of traditional agarose gels have been overcome, and several advantages over polyacrylamide gels have been demonstrated. The system is capable of high-resolution separation of small proteins and has a dynamic separation range equivalent to a 4%-20% gradient polyacrylamide gel. Furthermore, the staining of protein bands by Coomassie Brilliant Blue is very uniform in this gel, and depending on the protein, higher detection sensitivity can be obtained compared to SDS polyacrylamide gels. In Western blotting, proteins are more efficiently transferred to the membrane from the agarose gel than from polyacrylamide gels. Finally, the exceptional stability of agarose allows for gels to be precast and stored for a year.     

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.     

The free solution mobility of DNA

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.     

Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte

A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.     

The electrophoretic separation of curved cisplatin-modified DNA fragments on polyacrylamide gels is dependent on the voltage gradient

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The parameters of the electric field do not affect separation of 'normal' DNA fragments of comparable length.     

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity.     

Examination of the polypeptides of hepatitis B surface antigen

When the polypeptides of hepatitis B surface antigen were examined by SDS-polyacrylamide gel electrophoresis under a variety of conditions, anomalous results were found to be due to (i) variable and at times incomplete dissociation of polypeptides after boiling with 1% SDS and reducing agent, (ii) reaggregation of solubilized material under certain electrophoretic conditions and during laboratory manipulations, and (iii) the variable presence of additional components in hepatitis B surface antigen prepared from certain individual donors. When these factors were taken into account, two major components were consistently identified by discontinuous buffer polyacrylamide gel electrophoresis, of apparent mol. wt. 60000 to 70000 and 12000 to 14000. However, in view of the demonstrated limitations of this technique in examining HBsAg polypeptides, alternative methods are necessary to confirm the true mol. wt. of the unique virus-specified amino acid sequence present.     

Immunoglobulin E, viral antibodies and obstructive lung disease in adults. The relation between antibody level in serum, lung function and non-specific bronchial reactivity

The electrophoretic mobilities of DNA molecules in three different molecular weight ladders were measured in polyacrylamide gels containing different acrylamide concentrations (%T) and cross-linker ratios (%C), cast and run in Trisacetate-EDTA (TAE) buffer. The apparent pore radius of each gel was estimated from Ferguson plots of the relative mobilities of each of the DNA molecules, using the mobility of the monomer fragment in each molecular weight ladder as the reference mobility. The effective size of each of the DNA molecules was estimated from its radius of gyration. The apparent gel pore radii calculated in this manner ranged from 21 nm in gels containing 10.5%T, 5%C to 200 nm in gels with 4.6%T, 2%C, similar to the values observed for polyacrylamide gels cast and run in Tris-borate-EDTA (TBE) buffer (Holmes and Stellwagen, Electrophoresis 1991, 12, 612-619). Hence, the effective pore size of polyacrylamide gels is essentially independent of whether the gels are cast and run in TAE or TBE buffer.     

Purification of C3b inactivator and demonstration of its two polypeptide chain structure

C3b inactivator (C3bINA) was isolated from plasma by sequential ammonium sulfate gradient solubilization, anion exchange chromatography, gel filtration, and repeat ammonium sulfate gradient solubilization. The final product was pure as assessed by alkaline disc gel electrophoresis, isoelectric focusing, and SDS polyacrylamide gel electrophoresis, and elicited a monospecific rabbit antiserum. The normal serum concentration of C3bINA was found to be 53 +/- 9 microgram/ml (mean +/- S.D.). Heterogeneity of purified C3bINA was apparent on alkaline disc gel electrophoresis and isoelectric focusing, but not with SDS polyacrylamide gel electrophoresis and thus is attributed to forms of C3bINA that differ in charge rather than in size. SDS polyacrylamide gel electrophoresis of unreduced, alkylated C3bINA yielded a single stained band with an apparent m.w. of 93,000, whereas the reduced protein demonstrated two bands of 55,000 and 42,000 m.w., thereby establishing a composition of two disulfide-linked polypeptide chains for C3bINA.     

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.     

Detection of isozymes of deoxyribonucleases I and II on electrophoresed gels with picogram sensitivity using SYBR Green I

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).     

Capillary electrophoretic separation of 1 to 10 kbp sized dsDNA using poly(ethylene oxide) solutions in the presence of electroosmotic counterflow

DNA fragments of 1 to 10 kbp in length were separated by capillary electrophoresis (CE), using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow. The technique requires filling the capillary with the polymer solution by means of electroosmotic flow (EOF). Separation times of 6-7 min in PEO solutions ranging from 0.3 to 8 x 10(6) Mr at 375 V/cm were sufficient to separate the 11 components of the dsDNA ladder (0.5 to 10 kbp) by size. The migration behavior of the double-stranded (ds)DNA fragments, interpreted by "Ferguson plot analysis", in the system is indistinguishable from that previously reported for capillary zone electrophoresis (CZE) in a polyacrylamide solution without EOF. Potential advantages of conducting CZE using polymer solutions in the presence of EOF are: (i) Possibility of long migration times on short columns; (ii) possibility of introducing relatively viscous, high Mr polymer solutions into narrow capillaries; (iii) possibility of establishing polymer concentration gradients in capillaries; (iv) possibility of concentrating the starting zone by balancing electrophoretic migration and electroosmotic transport.     

Cytosine arabinoside induces apoptosis in cerebellar neurons in culture

Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC50 of approximately 60 microM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 microM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.     

An in vivo method for studying afferent fibre activity from cervical paravertebral tissue during vertebral motion in anaesthetised cats

PURPOSE: The effect of ethanol and formate radicals on the major proteins of human erythrocyte membranes has been investigated. MATERIALS AND METHODS: Human erythrocyte ghosts and of erythrocyte ghosts stripped of peripheric proteins were irradiated in phosphate buffer with 100 mmol dm(-3) ethanol or 100 mmol dm(-3) formate under N2 or N2O. The alterations of the proteins were investigated by SDS-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. RESULTS: In contrast to previous results on ribonuclease and on serum albumin the ethanol radicals were found to have a higher efficiency to damage erythrocyte membrane proteins than the formate radicals. Spectrin (Bands 1 and 2) and capnophorin (Band 3) showed the highest radiation-induced loss of all membrane proteins. When cysteamine or dithiothreitol were added to the erythrocyte ghosts with a similar OH-scavenging capacity as ethanol or formate, no degradation or aggregation of the membrane proteins could be observed even after a dose as high as 1800 Gy. CONCLUSIONS: The results of this study confirm the high radiosensitivity of spectrin and capnophorin to primary radicals. Similarly to soluble proteins, membrane-associated proteins are more significantly damaged by ethanol radicals than by formate radicals.