Showing papers on "Gel electrophoresis published in 1993"

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Abstract: We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.

Abstract: This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.

Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

Abstract: A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

The single cell gel electrophoresis assay (comet assay): A European review

Abstract: The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA

Abstract: The endogenous production of oxidative damage in DNA by free radicals released as a by-product of respiration is a likely cause of mutations which, if they occur in appropriate genes, may lead to cancer. Using an endonuclease specific for oxidized pyrimidines, in conjunction with the highly sensitive method of single cell gel electrophoresis, we have detected significant oxidative damage in untreated, freshly isolated lymphocytes from normal, healthy individuals.

Handbook of Capillary Electrophoresis

Abstract: ModesIntroduction to Capillary Electrophoresis, R.P. Oda and J.P. LandersMicellar Electrokinetic Chromatography, J.R. MazzeoCapillary Electrophoresis Separation of Enantiomers by Cyclodextrin Array Chiral Analysis, A. GuttmanCapillary Isoelectric Focusing, R. Rodr guez-D az, T. Wehr, M. Zhu, and V. LeviTheory and Practice of Capillary Electrochromatography, M.M. Dittmann and G.P. RozingAnalyteCapillary Ion Electrophoresis, W.R. JonesAnalysis of Small Organic Molecules by Capillary Electrophoresis, K.D. AltriaCapillary Electrophoresis of Peptides, T. van de Goor, A. Apffel, J. Chakel, and W. HancockCapillary Electrophoresis of Proteins, T. Pritchett and F.A. RobeyCarbohydrate Analysis by Capillary Electrophoresis, J.D. Olechno and J.A. NolanSeparation of DNA by Capillary Electrophoresis, K.J. Ulfelder and B.R. McCordEssential Aspects of Capillary ElectrophoresisOptical Detection Techniques for Capillary Electrophoresis, S.L. Pentoney, Jr. and J.V. SweedlerElectrochemical Detection in Capillary Electrophoresis, C. HaberData Analysis in Capillary Electrophoresis, B.J. WandersEffects of Sample Matrix on Capillary Electrophoretic Analysis, Z.K. ShihabiOn-Line Sample Preconcentration for Capillary Electrophoresis, D.S. Burgi and R.-L. ChienApplicationsCapillary Electrophoresis for the Analysis of Single Cells: Electrochemical, Mass Spectrometric, and Radiochemical Detection, F.D. Swanek, S.S. Ferris, and A.G. EwingCapillary Electrophoresis for the Analysis of Single Cells: Laser-Induced Fluorescence Detection, S.J. Lillard and E.S. YeungCapillary Gel Electrophoresis for Large Scale DNA Sequencing: Separation and Detection, N.J. DovichiCapillary Electrophoresis for the Analysis of Drugs in Biological Fluids, R.P. Oda, M.E. Roche, J.P. Landers, and Z.K. ShihabiUse of Capillary Electrophoresis for Binding Studies, F.A. RobeyImmunoassays and Enzyme Assays Using Capillary Electrophoresis, N.M. Schultz, L. Tao, D.J. Rose, Jr., and R.T. KennedyClinical Applications of Capillary Electrophoresis, R.P. Oda, V.J. Bush, and J.P. LandersSpecialized Aspects of Capillary ElectrophoresisCapillary Surface Modification in Capillary Electrophoresis, A.M. Dougherty, N. Cooke, and P. ShiehImproved Capillary Electrophoretic Separations Associated with Controlling Electroosmotic Flow, C.S. LeeContinuous Separations by Electrophoresis in Rectangular Channels, P.F. Gavin and A.G. EwingTwo-Dimensional Liquid Chromatography-Capillary Electrophoresis, D.J. Jeffery, T.F. Hooker, and J.W. JorgensonCapillary Electrophoresis-Mass Spectrometry, J.C. Severs and R.D. SmithMicrofabricated Devices for Performing Capillary Electrophoresis, S.C. Jacobson and J.M. RamseyFraction Collection with Micro-Preparative Capillary Electrophoresis, M.A. Strausbauch and P.J. WettsteinAppendix 1: Calculations for Practical UseAppendix 2: TroubleshootingAppendix 3: Seperation Conditions for Classes of AnalytesIndex

Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes

Abstract: Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.

A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin.

Abstract: A rapid, simple and reliable sex test that entails PCR amplification of a segment of the X-Y homologous gene amelogenin has been developed. We used a single pair of primers spanning part of the first intron which generated 106-bp and 112-bp PCR products from the X and Y homologues, respectively, that can be analyzed simply by agarose gel electrophoresis. Less than 1 ng of template DNA is required for gender assignment, and the test has been automated by the fluorescent tagging of the PCR products that are then quantitated during electrophoresis by automated fluorescence-detection technology. Quantitation enables sex chromosome aneuploidy to be determined, and the amelogenin intron sequence can also be co-amplified with several highly polymorphic microsatellite loci, thereby providing a combined gender/identity DNA test.

Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping.

Abstract: A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed. Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel. The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length. Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis. Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

Abstract: In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation

Abstract: A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or “programmed cell death”. This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [α 32P]-to the 3′-of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. © 1993 Wiley-Liss, Inc.

Annexin II is a major component of fusogenic endosomal vesicles.

Abstract: We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.

DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection.

Abstract: Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.

Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.

Abstract: HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

Detection of the initial stages of DNA fragmentation in apoptosis.

Abstract: DNA fragmentation during apoptosis proceeds through an ordered series of stages commencing with the production of DNA fragments of 300 kbp, which are then degraded to fragments of 50 kbp. The 50-kbp fragments are further degraded, in some but not all cells, to smaller fragments (10-40 kbp) and release the small oligonucleosome fragments that are recognized as the characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of the initial stages of DNA fragmentation using pulsed-field gel electrophoresis or a combination of pulsed-field gel electrophoresis and conventional agarose gel electrophoresis that allows detection of the DNA ladder in the same sample. A new method for the detection of high molecular weight DNA fragments on conventional agarose gels is presented, together with a rationale for the analysis of DNA fragmentation during apoptosis.

Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain.

Abstract: We have studied whether the delayed cell death induced by transient forebrain ischemia is associated with an inter-nucleosomal cleavage of DNA into oligonucleosome-sized fragments. The integrity of genomic DNA in various brain regions after a 20-min four-vessel ischemia was examined using gel elec-trophoresis. We found typical ladders of oligonucleosomal DNA fragments in the striatum and in the Ammon's horn. In the latter we also often found a random DNA degradation as a smear pattern. These findings were reinforced by a specific in situ labeling of DNA breaks in tissue sections. A dark staining of nuclei was observed in the cell bodies of neurons—in particular in the head of the caudate and in the vulnerable CAl hippocampal area. With biochemical and histological approaches, there was no evidence of DNA degradation in regions that are resistant to the injury. We conclude that the association of multiple mechanisms of cell damage may occur after a global ischemia. The regional variability in DNA fragmentation stresses the importance of using histological approaches in parallel with gel electrophoresis.

Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori.

Abstract: Gene hpaA, which codes for the receptor-binding subunit of the N-acetylneuraminyllactose-binding fibrillar hemagglutinin (NLBH) of Helicobacter pylori, was cloned and sequenced. The protein expressed by hpaA, designated HpaA, was identified as the adhesin subunit on the basis of its fetuin-binding activity and its reactivity with a polyclonal, monospecific rabbit serum prepared against NLBH purified from H. pylori. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blots (immunoblots) showed that the cloned adhesin has the same molecular weight (20,000) as that found on H. pylori. Also, HpaA contains a short sequence of amino acids (KRTIQK) which are all either identical or functionally similar to those which compose the sialic acid-binding motif of Escherichia coli SfaS, K99, and CFA/I. Affinity-purified antibody specific for a 12-residue synthetic peptide that included this sequence blocked the hemagglutinating activity of H. pylori and was shown by immuno-gold electron microscopy to react with almost transparent material on unstained H. pylori cells, which is consistent with previous observations concerning the location and morphology of the NLBH.

Islet Cell DNA Is a Target of Inflammatory Attack by Nitric Oxide

Abstract: NO has been identified recently as the prime islet-toxic product of inflammatory macrophages. The adverse effects of IL-1 on isolated islets also have been reported to involve NO. We now show that exposure of an islet cell suspension to the NO donor nitroprusside or to activated macrophages leads to DNA strand breaks. Macrophages did not induce DNA damage in the presence of the NO synthase inhibitor NG-methyl-L-arginine. DNA strand breaks were demonstrated at the level of single cells by a modified nick-translation procedure and confirmed by analysis of DNA fragmentation by gel electrophoresis. DNA strand breaks occurred within 1 h and preceded islet cell lysis. DNA damage could not be prevented by inhibitors of endogenous endonucleases. We conclude that islet cell DNA is an early target of NO action.

Polyvinylpyrrolidone-Agarose Gel Electrophoresis Purification of Polymerase Chain Reaction-Amplifiable DNA from Soils

Abstract: This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil This modification is to add polyvinylpyrrolidone to the agarose gel The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids

Ribonuclease Protection Assay

Abstract: Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.

Intracellular and extracellular processing of chromogranin A: Determination of cleavage sites

Abstract: Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a PI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and Cterminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity : pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65 -76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.

Identification of the soybean allergenic protein, Gly m Bd 30K, with the soybean seed 34-kDa oil-body-associated protein.

Abstract: The soybean allergenic protein, Gly m Bd 30K [Ogawa et al., J. Nutr. Sci. Vitaminol., 37, 555-565 (1991)] which is most strongly and frequently recognized by the IgE antibodies in sera of soybean-sensitive patients with atopic dermatitis, has been characterized. The allergen was isolated from the crude 7S-globulin fraction as an oligomeric form with a molecular weight of more than 3000,000 by gel-filtration chromatography. On two-dimensional gel electrophoresis, the native oligomeric allergen had an isoelectric point of about pH 4.5 and was dissociated into a monomeric form with a molecular weight of about 32,000 by the treatment with sodium dodecyl sulfate and 2-mercaptoethanol. The monomeric allergen had an N-terminal amino acid sequence and amino acid composition identical with those of the soybean seed 34-kDa oil-body-associated protein or the soybean vacuolar protein P34 with close homology to papain-like thiol proteinases [Kalinski et al., J. Biol. Chem., 267, 12068 (1992)]. The identity was further confirmed by the immunological cross-reactivity to the antibodies produced against each of the purified allargen and the 34-kDa oil-body-associated protein. By this observation, Gly m Bd 30K was shown to have about 30% sequence homology with Der pI, a house dust mite allergen that is a thiol proteinase from Dermatophagoides pteronyssius.

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Abstract: Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.

Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus.

Abstract: Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best method of typing methicillin-resistant S. aureus strains. Images