DNA Restriction Fragment Analysis Research Articles

Survival of Burkholderia pseudomallei in distilled water for 16 years

SummaryBurkholderia pseudomallei was examined after being maintained in distilled water at 25 °C for 16 years. The Gram stain was atypical (pale pink cocci or coccobacilli). The estimated number of live and dead B. pseudomallei was 3.8 × 107 cells/ml and 1.4 × 105 cells/ml, respectively. A colony count on agar of 1.0 × 106 cfu/ml suggested that a proportion of cells were in a viable but non-culturable state. Colony morphology was different from the parental isolate for 84% of colonies. Pulsed-field gel electrophoresis analysis of AvrII DNA restriction fragments revealed six different but related banding patterns, which may represent genomic rearrangement.

The use of selected starter Saccharomyces cerevisiae strains to produce traditional and industrial cachaça: a comparative study

Six Saccharomyces cerevisiae strains from cachaca fermentation were characterized for biomass, ethanol, glycerol, and acetic acid yields, as well as productivity. Three strains presenting the best fermentation parameters were selected for cachaca production. The experiments were carried out in an industrial distillery that distills this beverage in a stainless steel column, and in a traditional distillery that uses copper alembic for distillation. The permanence of the selected strains was studied by restriction fragment analysis of mitochondrial DNA. Strains UFMG-A1007 and UFMG-A2097 were prevalent in the vats during the 5 days of the fermentation period. Non-Saccharomyces strains were isolated during the entire fermentation period. In general, the cachacas produced in the stainless steel column had the highest concentrations of volatile acidity, acetaldehyde, esters, and higher alcohols. Both cachacas did not differ statistically in aroma, taste, and overall impression. The use of these indigenous S. cerevisiae strains as starter ferment could improve the sensory attributes of both industrial and traditional cachacas.

Spatial Variation in Bacterial DNA Profiles for Forensic Soil Comparisons

ABSTRACTThe feasibility of using bacterial DNA profiling of soil in forensic investigations is evaluated. Terminal restriction fragment (TRF) analysis of 16S ribosomal DNA may be employed to generate DNA profiles for use to discriminate between soil samples. To assess the localisation of the profiles and strengthen any evidential value of TRF analysis, small-scale spatial variability has been examined. Soil samples were collected at intervals along 2 m horizontal transects of three different ecosystems—a field, a forest, and a dune system—and TRF profiles compared. 13% to 20% of TRFs were common to all sampling points, for all transects. The mean similarity for TRF profiles at all sampling points ranged from 49.3% for the dune system to 58.4% for the field transect. ‘Patch size’ (distance along the transect in which DNA profiles showed highest similarity) ranged from 20–80 cm for each ecosystem. This small- scale variability may cause problems if forensic ‘matching’ of soil DNA profiles is attempted, unless the precise location from which the soil sample was originally taken could be identified. The implications of these findings for forensic fingerprinting of soil samples are discussed.

Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes

Abstract. This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA) particles in the atmosphere. Samples of fine particulate matter (PM2.5) and total suspended particulates (TSP) have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze). From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP) were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses. Mass fractions of DNA in PM2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ~7 ng m−3, indicating that human adults may inhale about one microgram of DNA per day (corresponding to ~108 haploid bacterial genomes or ~105 haploid human genomes, respectively). Most of the bacterial sequences found in PM2.5 were from Proteobacteria (42) and some from Actinobacteria (10) and Firmicutes (1). The fungal sequences were characteristic for Ascomycota (3) and Basidiomycota (1), which are known to actively discharge spores into the atmosphere. The plant sequences could be attributed to green plants (2) and moss spores (2), while animal DNA was found only for one unicellular eukaryote (protist). Over 80% of the 53 bacterial sequences could be matched to one of the 19 T-RF peaks found in the PM2.5 samples, but only 40% of the T-RF peaks did correspond to one of the detected bacterial sequences. The results demonstrate that the T-RFLP analysis covered more of the bacterial diversity than the sequence analysis. Shannon-Weaver indices calculated from both sequence and T-RFLP data indicate that the bacterial diversity in the rural samples was higher than in the urban and alpine samples. Two of the bacterial sequences (Gammaproteobacteria) and five of the T-RF peaks were found at all sampling locations.

Assessing the Potential of Bacterial DNA Profiling for Forensic Soil Comparisons*

A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.

Emergence and prevalence of β-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan

Forty-six cephem-resistant Klebsiella pneumoniae strains with minimum inhibitory concentrations >8 μg/mL for cefpodoxime and cefmetazole were selected from clinical isolates obtained between 2000 and 2002 from eight hospitals on Northern Kyushu Island, Japan. We investigated the mechanisms of resistance to cephems in these 46 K. pneumoniae isolates. The results of isoelectric focusing of β-lactamases produced by these isolates, polymerase chain reaction for detection of various Class A, Class B and Class C β-lactamases, and determination of the sequence of the β-lactamase structural gene showed that most of these isolates had various types of broad-spectrum β-lactamases. Of the 46 isolates, 2 were CMY-2 β-lactamase producers and 41 were DHA-1 β-lactamase producers. Forty of the 41 DHA-1 β-lactamase producers simultaneously produced SHV-12 extended-spectrum β-lactamase (ESBL), and the remaining isolate simultaneously produced SHV-27. Furthermore, one DHA-1 and SHV-12 β-lactamase producer also produced IMP-1 β-lactamase. The only broad-spectrum β-lactamase with another isolate was IMP-1. Chromosomal DNA restriction fragment analysis using XbaI suggested that nosocomial infection due to DHA-1 and SHV-12 β-lactamase producers had occurred at two centres. This is the first report of nosocomial infection due to DHA-1 β-lactamase-producing K. pneumoniae including other plasmid-encoded AmpC β-lactamases in Japan. The mechanisms of resistance of 44 of the 46 isolates to cephalosporins and cephamycins were ESBL production and/or plasmid-encoded AmpC β-lactamase and/or IMP-1 β-lactamase production. For two isolates, the mechanism of resistance to could not be identified. These results show that it is necessary to minimise the prevalence of these resistant strains as it will be a very serious problem if organisms producing these broad-spectrum β-lactamases increase in clinical situations. It is important to detect these strains sooner and to perform rigorous infection control earlier.

Two families with nonsyndromic low-frequency hearing loss harbor novel mutations in Wolfram syndrome gene 1

Although hereditary hearing loss is highly heterogeneous, only a few loci have been implicated with low-frequency hearing loss. Mutations in one single gene, Wolfram syndrome 1 (WFS1), have been reported to account for most familial cases with this type of hearing impairment. This study was conducted to determine the cause of nonsyndromic low-frequency hereditary hearing impairment in two large families. Two large families from Switzerland and United States with low-frequency hearing loss were identified. Genomewide linkage analysis was performed followed by mutation screening in the candidate gene WFS1 with direct DNA sequencing and restriction fragment analysis. Both families were linked to DFNA6/14/38 with lod scores>3. Two novel heterozygous missense mutations in WFS1 were identified: c.2311G>C leading to p.D771H in the Swiss family and c.2576G>C leading to p.R859P in the US family. The sequence alteration was absent in 100 control chromosomes. Nonsyndromic low-frequency hereditary hearing impairment seems to be predominantly a monogenic disorder due to WFS1. We confirm that most mutations in WFS1 associated with isolated low-frequency hearing loss are clustered in the C-terminal protein domain coded by exon 8.

Intramolecular recombination and deletions in mitochondrial DNA of senescent, a nuclear-gene mutant of Neurospora crassa exhibiting “death” phenotype

In Neurospora crassa, a nuclear-gene mutant, senescent, derived from a phenotypically normal wild isolate of Neurospora intermedia exhibits a ‘death’ phenotype. Regardless of the composition of the culture medium, the mycelium ceases to grow in 2–6 subcultures at 26 °C and 1 or 2 subcultures at 34 °C. Senescence of vegetative mycelium is associated with deficiencies in cytochromes aa 3 and b and reduced oxygen uptake. The restriction fragment analysis of mitochondrial DNA from senescing mycelia showed deletions and gross sequence rearrangements. Analysis of mitochondrial DNA of ( sen + sen +) heterokaryons constructed with “excess” sen cytoplasm suggested correlation between mtDNA deletions and senescence. Three novel sen-specific EcoRI fragments of sizes 3.6, 3.9, and 4.4 kb were cloned, sequenced, and analyzed. Nucleotide sequences of the sen-specific EcoRI fragments suggested that deletions were a consequence of intramolecular recombination between EcoRI-5 and -10 and/or between EcoRI-8 and -10. The recombination junctions were close to stretches of GC-rich- PstI palindromic sequences that potentially form stable hairpin structures and might facilitate recombination between homologous repeats as short as 6–10 bp. These observations suggest that the wild-type ( sen +) allele encodes a factor that protects the mitochondrial genome from undergoing intramolecular recombination and deletions. In this respect sen + (linkage group V) has a function similar to nd + (linkage group I) and the two gene products probably have mutually exclusive roles in suppressing cruciform-associated and homologous recombination, respectively, thus safeguarding mitochondrial genome integrity. The sen + allele most likely codes for a factor involved in recombination, repair or replication of the mitochondrial genome, or a transcription factor that regulates the expression of genes affiliated with mitochondrial DNA metabolism.

Genomic analysis of penicillin-resistant Streptococcus pneumoniae in Southeastern Michigan.

The emergence of multidrug resistance within Streptococcus pneumoniae population was analysed, correlating penicillin resistance Pen(R) with secondary antibiotic resistance, capsular serotype, and genetic diversity among isolates. DNA fingerprinting, following macro-restriction enzyme digestion and pulse field gel electrophoresis (PFGE), and restriction fragment analysis of the PBP 2b gene, following PCR amplification, were performed on the Pen(R) S. pneumoniae, among 377 clinical isolates obtained from the clinical microbiology laboratory (University of Michigan Medical Center). Overall 35% of the isolates were Pen(R) of which 45% demonstrated high-level penicillin (Pen(R)-R, MIC>1). Respiratory isolates were more likely to be Pen(R) (p <0.001) than non-respiratory isolates and the rate of Pen(R)-R was significantly increased in children <10 years of age (59.6%, p <0.02). Secondary antibiotic resistance was more frequently associated with Pen(R)-R. Genomic DNA fingerprinting analysis and restriction fragment analysis of the PBP 2b gene demonstrated genomic divergence with discrete conserved pattern in the PBP 2b gene among the resistant isolates. The emergence of multidrug resistance in the S. pneumoniae population in SE Michigan is not due to expansion of a single or limited number of resistant clones, is occurring most frequently in the paediatric population and is associated with a decreased susceptibility to penicillin.

Mono- and bis-intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis.

The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.

Mono- and bis-intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

In silico analysis of the restriction fragments length distribution in the human genome

The Restriction On Computer (ROC) program (freely available at http://www.mcb.harvard.edu/gilbert/ROC) was developed and used to analyze the restriction fragment length distribution in the human genome. In contrast to other programs searching for restriction sites, ROC simultaneously analyzes several long nucleotide sequences, such as the entire genomes, and in essence simulates electrophoretic analysis of DNA restriction fragments. In addition, this program extracts and analyzes DNA repeats that account for peaks in the restriction fragment length distribution. The ROC analysis data are consistent with the experimental data obtained via in vitro restriction enzyme analysis (taxonomic printing). A difference between the in vitro and in silico results is explained by underrepresentation of tandem DNA repeats in genomic databases. The ROC analysis of individual genome fragments elucidated the nature of several DNA markers, which were earlier revealed by taxonomic printing, and showed that L1 and Alu repeats are nonrandomly distributed in various chromosomes. Another advantage is that the ROC procedure makes it possible to analyze the nonrandom character of a genomic distribution of short DNA sequences. The ROC analysis showed that a low poly(G) frequency is characteristic of the entire human genome, rather than of only coding sequences. The method was proposed for a more complex in silico analysis of the genome. For instance, it is possible to simulate DNA restriction together with blot hybridization and then to analyze the nature of markers revealed.

Microcapillary electrophoresis devices fabricated using polymeric substrates and X-ray lithography

An integrated microdevice for the analysis of DNA restriction fragments or sequencing fragments is currently being constructed in our laboratory and consists of two principal components: a piezo-driven micropump and a microelectrophoresis device with integrated fluorescence detector. The syringe pump consisted of a piezoelectric actuator and a pivoted lever for amplification to deliver solvents free from pump pulsations at volumetric flow rates approaching 1 nL/min, even at high loading levels (high output pressures). The flow was programmed by controlling the voltage waveform to the piezo-actuator to produce a linear displacement of 80 μm and, by using the pivoted lever, a total linear displacement of 650 μm was achieved. The total volume delivered in a single pump stroke was 565 nL. The piezo-pump was found to adequately deliver stable flow of solutions with loading pressures as high as 3.79×105 Pa (actual loading pressure at the piezo is 3.41×106 Pa). The second component consisted of an electrophoresis device micromachined in polymethylmethacrylate (PMMA) using X-ray lithography (LIGA). The device was fabricated using a transfer mask technique, in which the channel topography was transferred to a PMMA substrate coated with a positive photoresist and a thin Au/Cr plating base using an optical mask with subsequent X-ray exposure to produce the desired channel topography. The channels were found to be 20 μm in width (determined by optical mask) with channel depths of 50 μm (determined by X-ray exposure time) and aspect ratios of approximately 1:10,000, significantly better than those obtained using wet-chemical etching in glass. The detection apparatus used a fiber optic to deliver the laser light to the electrophoresis device with the emission collected via the same fiber and the wavelength sorting accomplished with a dichroic filter. Since the electroosmotic flow in the PMMA was found to be approximately 5 times smaller compared to glass at typical separation pH for DNA (8.6), the walls of the PMMA device would not require a polymer coating to reduce this flow when performing high-resolution DNA separations. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 413–422, 1998

DNAフラグメント解析からみたnivalenol産生性Fusarivm sp.Fn 2Bの化学分類

DNAフラグメント解析からみたnivalenol産生性Fusarivm sp.Fn 2Bの化学分類

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to ‘fingerprint’ accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.

Integrated Capillary Electrophoresis on Flexible Silicone Microdevices: Analysis of DNA Restriction Fragments and Detection of Single DNA Molecules on Microchips

Microchips for integrated capillary electrophoresis systems were produced by molding a poly(dimethylsiloxane) (PDMS) silicone elastomer against a microfabricated master. The good adhesion of the PDMS devices on clean planar surfaces allows for a simple and inexpensive generation of networks of sealed microchannels, thus removing the constraints of elaborate bonding procedures. The performance of the devices is demonstrated with both fast separations of φX-174/HaeIII DNA restriction fragments labeled with the intercalating dye YOYO-1 and fluorescently labeled peptides. Detection limits in the order of a few zeptomoles (10(-)(21) mol) have been achieved for each injected DNA fragment, corresponding to a mass detection limit of ∼2 fg for the 603 base pair fragment. Single λ-DNA molecules intercalated with YOYO-1 at a base pair-to-dye ratio of 10:1 could be detected with an uncomplicated laser-induced fluorescence detection setup. High single-molecule detection efficiency (>50%) was achieved under electrophoretically controlled mass transport conditions in PDMS microchannels.

Distribution of parental DNA markers inEncelia virginensis (Asteraceae: Heliantheae), a diploid species of putative hybrid origin

Morphological, geographical and ecological evidence suggests thatEncelia virginensis is a true-breeding diploid species derived from hybrids ofE. actoni andE. frutescens. To test this hypothesis, we examined the chloroplast and nuclear DNA of severalEncelia species. PCR amplification targeted three separate regions of chloroplast DNA:trnK-2621/trnK-11,rbcL/ORF106, andpsbA3/TrnI-51, which amplify 2600bp, 3300bp and 3200bp fragments respectively. Restriction fragment analysis of chloroplast DNA revealed no variation that could be used to discriminate between the parent species. A RAPD analysis using 109 dekamer primers was used to analyze the nuclear genome.Encelia actoni andE. frutescens were distinguished by several high-frequency RAPD markers. In populations ofE. virginensis, these markers were detected in varying proportions, and no unique markers were found. Evidence from the nuclear genome supports the hypothesis thatE. virginensis is of hybrid origin. ThatE. virginensis may have arisen by normal divergent speciation followed by later introgression remains a possibility, however, and is not formally ruled out here. Diploid hybrid speciation inEncelia differs from other documented cases in that there are no discernible chromosome differences between the species, and all interspecific hybrids are fully fertile. In addition, apparent ecological selection against backcross progeny provides an external barrier to reproduction between F1 progeny and the parental species. These characteristics suggest that hybrid speciation inEncelia may represent an alternative model for homoploid hybrid speciation involving external reproductive barriers. In particular, this may be the case for other proposed diploid hybrid taxa that also exhibit little chromosomal differentiation and have fertile F1s.

Differentiation of Staphylococcus xylosus Strains from Italian Sausages by Antibiotyping and Low Frequency Restriction Fragment Analysis of Genomic DNA

The aim of this study was to identify the strengths and weaknesses of two typing methods, antibiogram typing and low-frequency restriction fragment analysis of genomic DNA, for the differentiation of Staphylococcus xylosus strains. Twenty-eight Staphylococcus xylosus strains isolated from Italian fermented sausages (Naples-type salami) and S. xylosus DSM 20266 (Type strain) and DSM 6179 were analysed. Antibiotyping, recorded on the basis of susceptibility testing of 16 antibiotics, allowed 21 antibiogram-types to be distinguished. Pulsed-field gel electrophoresis techniques combined with DNA digestion by infrequently cutting enzymes such as Sma I produced 22 pattern-types among the 30 strains of S. xylosus analysed. The association of antibiotyping and PFGE typing clearly distributed S. xylosus strains into 30 combinations, showing that all the strains can be differentiated. Thus, the combination of these two techniques, in spite of limitations due to instability of a phenotype-based system, actually turned out to be the most reliable methodological approach for differentiating strains of S. xylosus.

Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis

Capillary electrophoresis has become a powerful analytical tool for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. The use of replaceable polymer solutions increases the lifetime of the capillary, improves the repeatability of the migration times and enables pressure injection. When pressure injection is used, rather than electrokinetic injection, it is assured that a representative part of the sample is introduced into the capillary. The possible lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent labels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denaturing conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increased selectivity compared to separation of that sample in its ds form. In this work, we exploited this increased selectivity for the analysis of denatured PCR products. It was found that DNA separated in the ss form yields superior separation; that is, a given analysis can be achieved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if high resolution, size dependent separation is required. The enhanced resolution achieved with DNA migrating in the ss form enabled the separation of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longer than 6 min.