High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Fluorescent properties of histone-1-anilinonaphthalene 8-sulfonate complexes in the presence of denaturant agents: application to the rapid staining of histones in urea and Triton-urea-polyacrylamide gels

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 micrograms of histone per band.     

Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.

A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).     

A DNA cycle sequencing reaction that minimizes compressions on automated fluorescent sequencers.

We recently demonstrated that most band compressions (>70%) on DNA sequencing gels result from the presence of a single sequence motif, 5'-YGN1-2AR-3', where Y and R indicate base-pairing pyrimidine and purine residues, respectively. This finding raised the possibility that the use of 7-deaza-dATP instead of dATP in chain termination sequencing reactions would resolve most of the band compressions. Thus, we examined the effects of 7-deaza-dATP on DNA sequencing using thermostable DNA polymerases. The results indicate that the replacement of dATP with 7-deaza-dATP in conventional cycle sequencing reactions can successfully eliminate most band compressions without sacrificing sequencing performance.     

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5′-base overhang. Regardless of the 5′-end structure, all bleomycin-induced DSBs possess 3′-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of ≤0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires ≤100 ng substrate DNA and ≥50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.     

Preliminary characterization of chelation-sensitive nucleoprotein particles

Nucleoprotein particles (B2), isolated following digestion of calf thymus chromatin with micrococcal nuclease, are resolved on a non-chelating Bio-Gel A-5m column. B2 protein electrophoresis showed the presence of several H1 species and several nonhistone proteins but was depleted in core histones. DNA electrophoresis demonstrated that native B2 DNA has a length of about 46 base pairs. On DNA sequencing gels, the length distribution of denatured B2 DNA ranged from 12 to 35 bases with a weighted average chain length of about 26 bases. Depletion of a 20 base band in B2 DNA suggested specific protection of internucleosomal DNA sites during the nuclease digestion.     

Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5.

Electrophoresis of the mixture of proteins from purified snRNPs U1, U2, U4/U6 and U5 on SDS-polyacrylamide gels that had been allowed to polymerise in the presence of high TEMED concentrations have revealed the presence of proteins in the snRNPs that previously had eluded detection. The most striking case is that of protein D, heretofore generally observed as a single broad band; in high-TEMED gels, this splits into three clearly-separated bands, identified as three distinct proteins. We have denoted these proteins D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa). Chemical and immunological studies have shown that D1 is identical with the common snRNP protein D, whose structure was recently resolved by cDNA cloning (Rokeach et al. (1988), Proc. Natl. Acad. Sci. USA, 85, 4832-4836) and that D2 and D3 are clearly distinct from D1 and very probably from each other. In addition to D1, proteins D2 and D3 are present in purified U1, U2, U4/U6 and U5 snRNPs isolated from HeLa cells, so these also belong to the group of common snRNP proteins. They are also found in snRNPs isolated from mouse cells, indicating that the role of these proteins in the structure and/or function of UsnRNPs has been conserved in evolution. Interestingly, patients with systemic lupus erythematosus produce populations of anti-Sm autoantibodies that react differentially with the D proteins; some recognise all of them and others only a subset. The high-TEMED gels allow improved resolution not only of the D proteins, but also of some of the U5-specific proteins contained in 20S U5 snRNPs, in particular the 15-kDa protein. In addition, under these conditions, the common G protein, previously observed as a single band, appears as a doublet. Whether the additional band represents a distinct common snRNP protein or a post-translationally modified version of G is not yet known.     

Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.     

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.     

Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.     

Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate

A silver stain technique using treatment with potassium permanganate for visualisation of proteins and DNA separated by gel electrophoresis was developed and applied both to the very thin (thickness 0.1-0.2 mm) and to the usual 1-2 mm thick gels. The technique is reproducible, only stable chemicals are used and it is shortened to about 1 h even for the 1 mm gel (30 min for the 0.2 mm gel). It is applicable to gels thicker than 2 mm with somewhat longer washes. Amounts as low as 2 X 10(-10) g of protein per band have been detected. Protein bands of different colours are obtained. The technique has been used with success in continuous, discontinuous and 2D-gel systems. Bands have been detected which were not observed when the other silver staining techniques were used.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.     

Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.

A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.     

Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry.

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repeating restriction fragments of human DNA.

Human DNA digested with Hae III showed multiple repeats of a 170 base pair fragment. The most prominent band was the 340 base pair dimer, estimated to be 0.8% of the entire genome. Eco R1 and Hha I yielded fragments with similar electrophoretic mobility to the Hae III dimer. In each case this band was markedly enriched in DNA reassociating at a 0t of less than or equal to 1. Hybridization of the Hae III dimer to gels eluted on to filters demonstrated that the multiple Hae III fragments and Eco R1 fragments contained compatible sequences. These sequences may comprise a distinct subclass of DNA.     

Spectrin band 1 and 2 are antigenically distinct and are not composed of subunits.

Human erythrocyte membranes and freshly isolated spectrin were separated into their constituent peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptides were electrophoresed from slices of such gels into agarose gels containing anti-spectrin antibodies and Triton X-100. In fresh preparations, precipitin arcs were observed only against peptides migrating as bands 1 and 2. It was found that bands 1 and 2 did not cross-react. There were two major arcs from band 1 and one principal arc from band 2, plus minor splitting of these arcs. None of the band 1 arcs fused with band 2 arcs. In fresh erythrocyte ghosts only bands 1 and 2 reacted with anti-spectrin; bands 2.1, 3, and 5, in particular, showed no precipitin arcs. However, in aged ghosts, arcs appeared in the band 3 region; in aged isolated spectrin, arcs appeared in the band 2.1 region; and in trypsin-degraded spectrin, reactive species occurred in all molecular weight classes. It is concluded that spectrin has no subunits smaller than 220,000 molecular weight and that bands 1 and 2 are immunochemically distinct.