Rapid DNA Analysis Research Articles

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P<0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.

The silicon that Moves and Feels Small Living Things

The silicon microelectronic chips that make today's computers possible are emerging as powerful tools for rapid and sensitive analysis of small biological objects, including cells, proteins, DNA, and viruses. Major new and exciting developments in the interface of solid-state circuits and biological entities are discussed in this article.

TaxMan: a taxonomic database manager.

BackgroundPhylogenetic analysis of large, multiple-gene datasets, assembled from public sequence databases, is rapidly becoming a popular way to approach difficult phylogenetic problems. Supermatrices (concatenated multiple sequence alignments of multiple genes) can yield more phylogenetic signal than individual genes. However, manually assembling such datasets for a large taxonomic group is time-consuming and error-prone. Additionally, sequence curation, alignment and assessment of the results of phylogenetic analysis are made particularly difficult by the potential for a given gene in a given species to be unrepresented, or to be represented by multiple or partial sequences. We have developed a software package, TaxMan, that largely automates the processes of sequence acquisition, consensus building, alignment and taxon selection to facilitate this type of phylogenetic study.ResultsTaxMan uses freely available tools to allow rapid assembly, storage and analysis of large, aligned DNA and protein sequence datasets for user-defined sets of species and genes. The user provides GenBank format files and a list of gene names and synonyms for the loci to analyse. Sequences are extracted from the GenBank files on the basis of annotation and sequence similarity. Consensus sequences are built automatically. Alignment is carried out (where possible, at the protein level) and aligned sequences are stored in a database. TaxMan can automatically determine the best subset of taxa to examine phylogeny at a given taxonomic level. By using the stored aligned sequences, large concatenated multiple sequence alignments can be generated rapidly for a subset and output in analysis-ready file formats. Trees resulting from phylogenetic analysis can be stored and compared with a reference taxonomy.ConclusionTaxMan allows rapid automated assembly of a multigene datasets of aligned sequences for large taxonomic groups. By extracting sequences on the basis of both annotation and BLAST similarity, it ensures that all available sequence data can be brought to bear on a phylogenetic problem, but remains fast enough to cope with many thousands of records. By automatically assisting in the selection of the best subset of taxa to address a particular phylogenetic problem, TaxMan greatly speeds up the process of generating multiple sequence alignments for phylogenetic analysis. Our results indicate that an automated phylogenetic workbench can be a useful tool when correctly guided by user knowledge.

An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 contains redundant tiling of sequences for 500 of the most common haplotypes including single-nucleotide changes, insertions, and deletions. Sequencing results from 14 primary head and neck tumor tissues demonstrated that the v2.0 MitoChips detected a larger number of variants than the original version. Multiple coding region variants detected only in the second generation MitoChips, but not the earlier chip version, were further confirmed with conventional sequencing. Moreover, 31 variations in noncoding region were identified using MitoChips v2.0. Replicate experiments demonstrated >99.99% reproducibility in the second generation MitoChip. In seven head and neck cancer samples with matched lymphocyte DNA, the MitoChip v2.0 detected at least one cancer-associated mitochondrial mutation in four (57%) samples. These results indicate that the second generation MitoChip is a high-throughput platform for identification of mitochondrial DNA mutations in primary tumors.

Ultrasonic Decalcification Offers New Perspectives for Rapid FISH, DNA, and RT-PCR Analysis in Bone Marrow Trephines

The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.

FIRST RECORD OF NANNOCHLOROPSIS LIMNETICA (EUSTIGMATOPHYCEAE) IN THE AUTOTROPHIC PICOPLANKTON FROM LAKE BAIKAL1

Three new strains of eukaryotic picoplankton, isolated from Lake Baikal, were identified as Nannochloropsis limnetica Krienitz, Hepperle, Stich &amp; Weiler. To date, N. limnetica had only been detected in small German and North American inland water bodies. On determination of the 18S rDNA sequence, the three new strains were found to be identical to each other and to the type strain KR 1998/3 (GenBank accession no. AF251496). Rapid analysis of polymorphic DNA–PCR revealed that the genotypes were different, although the Baikalian eustigmatophycean strains were more similar to each other compared with the type strain KR 1998/3 from Germany. Ecophysiological differences were also evident between the new strains from Lake Baikal and the type strain from growth rate determinations. The morphological characteristics were similar to that of a previous description of the species. However, although the cells of Eustigmatophyceae usually propagate by autosporulation, in these newly detected species germination of single daughter cells from thick‐walled cells was observed for the first time. Based on pigment analysis, the occurrence of Eustigmatophyceae in Lake Baikal was estimated. Eustigmatophyceae were established to be common members of the phytoplankton community of this large oligotrophic Siberian lake and occurred throughout the year, even under the ice cover during winter. Moreover, they peaked during early summer and in the South Basin. Hence, the widely accepted opinion that Chlorophyceae solely comprise the eukaryotic picoplankton should be changed and the Eustigmatophyceae considered.

Rapid Quantitative Analysis of Human Cytomegalovirus DNA by the Real-Time Polymerase Chain Reaction Method

Human cytomegalovirus (CMV) infection is a progressive and life-threatening complication in immunocompromised patients even now. Therefore, early and accurate treatment based on rapid and certain detection is needed to prevent fatal CMV infection diseases. To study a quicker, simpler, and less expensive method of quantitative analysis using real-time polymerase chain reaction based on the SYBR Green I method of CMV detection for appropriate treatment of CMV infection in immunocompromised patients. We quantified 50 samples tested by direct immunoperoxidase staining of leukocytes with peroxidase-labeled monoclonal antibody (C7-HRP test), 30 samples from healthy persons, and 47 samples from 7 patients suspected of having CMV infection diseases. We used the primer set in the pp65 gene of CMV and whole blood without a preparatory process. The setting for the study was the First Department of Pathology, Kurume University School of Medicine, St Mary's Hospital, and the Gene Section of the Clinical Laboratory at St Mary's Hospital, Fukuoka, Japan. The results obtained with this method corresponded well with conventional C7-HRP tests and demonstrated excellent reproduction. Additionally, the results were better correlated with the clinical course than were C7-HRP tests. This method was more useful than the C7-HRP test as a rapid diagnostic test for early treatment of CMV infection. This test also demonstrated its usefulness for monitoring CMV infection during treatment using ganciclovir. Moreover, it was quicker, simpler, and cheaper than other real-time polymerase chain reaction methods.

Dissection and Recovery of Chromosome Fragments by Atomic Force Microscopy

DNA analysis of specific regions in chromosome is important for acquirement of valuable genes and study of genetic diseases. Recently, atomic force microscopy (AFM) has also been applied to the chromosome dissection and the recovery of the chromosome fragments. However, any recovery method with high reproducibility by AFM has not been established yet. We developed a highly reproducible dissection and recovery method for chromosome fragments by using AFM, named “scratch method”. This method was available for different sized chromosomes both silkworm chromosome in pachytene phase and human metaphase chromosome of somatic cell. The scratch method will contribute to a development of more efficient and rapid DNA analysis in future.

Microsatellite loci for Myrmica kotokui and their application in some congeneric ant species distributed in northern Japan

AbstractWe developed seven polymorphic microsatellite loci for Myrmica kotokui from RAPD (rapid analysis of polymorphic DNA) fragments. These loci showed two to six alleles with expected and observed heterozygosity of 0.13–0.73 and 0.14–0.78, respectively. These loci are transferable to the congeneric species as Myrmica rubra, Myrmica luteola and Myrmica taediosa from northern Japan. These loci will allow analyses of genetic structure of Myrmica species at both the colony level and population levels.

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis, Fabaceae)

AbstractThis work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.

DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures.

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80 degrees C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N-dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDEA30). DNA-polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70 degrees -75 degrees C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50 degrees C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60 degrees C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50 degrees C. Molecular masses of these polymers were determined by tandem gel permeation chromatography-multiangle laser light scattering method (GPC-MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.

SNP analysis by allele-specific extension in a micromachined filter chamber.

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.

Sequences of E. coli O157:H7 detected by a PCR-acoustic wave sensor combination.

In this paper, we report on the amplification by polymerase chain reaction (PCR) of a 509 base sequence unique to E. coli O157:H7. Immobilization of a probe for the bacterium on an acoustic wave sensor by the biotin-neutravidin interaction was employed to detect the on-line hybridization of the sequence with the sample obtained from PCR. The limit of detection was found to be 10(-8) M, which suggests that the method may be useful for detecting the organism in food, water and clinical samples. Finally, this approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.

Development and application of a sensitive and rapid immunoassay for the quantitation of N7-methyldeoxyguanosine in DNA samples.

N7-Methyldeoxyguanosine (N7-MedG) in DNA is a biomarker of exposure to environmental and endogenous methylating agents and may be of use in epidemiological studies. To quantitate N7-MedG in human samples, a sensitive assay system that uses only small quantities of DNA (<10 microg) is required. To this end, polyclonal antibodies against the imidazole ring-opened form of N7-MedG have been used to develop a highly sensitive immunoslot blot (ISB) assay. The limit of detection of the assay is 0.10 micromol of N7-MedG/mol of deoxyguanosine (dG) using 1 microg of DNA per analysis. The method was optimized using in vitro-methylated calf thymus DNA and then applied to a study of DNA methylation in liver and brain tissues of mice following a single iv dose of the antitumor agent Temozolomide. The amount of N7-MedG in both tissues was strictly proportional to dose over a range of 10-200 mg of Temozolomide/kg of body weight. The ISB assay was then validated using pyloric DNA of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and DNA samples from human bladder tumors, for both of which N7-MedG levels had already been quantitated by an HPLC/(32)P-postlabeling method previously described. The results showed a high degree of correlation (r = 0.98) between the two assays. The ISB assay was then applied to a range of human samples. A series of peripheral blood mononuclear cell DNA samples from cancer patients following treatment with Temozolomide had levels of N7-MedG ranging from 0.22 to 320 micromol/mol of dG. DNA samples from colon carcinoma and normal colorectal mucosa from individuals not known to be exposed to methylating agents contained levels of 0.11-1.34 micromol of N7-MedG/mol of dG. The ISB assay offers the potential for the rapid and high-throughput analysis of DNA obtained from routine biopsies and blood samples, thus enabling the determination of the extent of human exposure to environmental and endogenous sources of methylating agents in large-scale biomonitoring studies.

Integrated system for rapid PCR-based DNA analysis in microfluidic devices.

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.

Application of PCR to Detect Varietal Purity in Barley Malt

The North American Barley Genome Mapping Project (NABGMP) generated several hundred molecular markers for mapping. Most of the NABGMP clones have been sequenced and sequence-tagged-site (STS) specific primers developed for polymerase chain reaction (PCR)-based analysis. We used the STS-PCR approach to identify amplified DNA fragments that differ in structure among genotypes. These primers, with the use of endonucleases, permit rapid analysis of DNA from young leaf tissue, harvested grain, or malted barley. Molecular markers were identified that differentiated cultivars within and between the two-row and six-row malting and feed barley germ plasm. We modified current DNA isolation protocols to have practical application in industry. Bulk DNA extractions were used to detect if an allotment of malt was contaminated with an offtype cultivar. The limit of resolution was as low as 1% for some cultivars using specific primer sets. Single malt-kernel extractions were used to quantify the mixture. These DNA isolation protocols and PCR-based techniques are rapid, relatively inexpensive, and more accurate and informative than grain morphology. The use of molecular techniques and DNA typing can be used to certify varietal purity and help ensure that finished malt, based on varietal performance, meets customer expectations.

PH-dependent specific binding and combing of DNA

Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed.

Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene.

Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radioactive probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient's other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent's phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity.

Analysis of microsatellite DNA using a non-radioactive method: blotting from polyacrylamide gel to nylon membrane using a gel dryer with subsequent hybridization to digoxigenin-labelled (dCdA)8 oligonucleotide probes

A non-radioactive method for the analysis of microsatellite DNA polymorphisms (mainly (CA)n-repeats) is described. Variation in length of (CA)n and (GT)n blocks was demonstrated directly by amplification of DNA from within and immediately flanking the repeat blocks using the polymerase chain reaction. The amplified DNA was resolved on a denaturing polyacrylamide gel, transfered to a nylon membrane using a gel dryer, and subsequently hybridized with digoxigenin-labelled (dCdA)8 oligonucleotide probes. This method enables easy and rapid analysis of polymorphic microsatellite DNA.

High speed polymerase chain reaction in constant flow.

A new simple reactor of the tubing type was developed for polymerase chain reaction (PCR). A thin Teflon capillary tube was used as a tubing reactor in which the reaction mixture of PCR was driven by a pump at a constant flow rate. The sample was treated with three successive thermal stages for denaturation, annealing, and elongation of DNA and primers as a function of the position in the tube. The amplification yield was about a half of that obtained by a commercial thermocycler. Moreover, the total reaction time from 12 to 18 min, which was one-tenth of the time generally required by conventional thermocyclers using metal blocks, assured substaintial amplification of a DNA fragment. In addition, this reactor could be also used for rapid cycle-sequences. This new device will be easily incorporated into automated and rapid DNA analysis systems for DNA sequencing.