DNA Sample Preparation Research Articles

Simultaneous Intraoperative Detection of Methicillin-Resistant Staphylococcus and Pan-Bacterial Infection During Revision Surgery

Periprosthetic infection is one of the most serious complications of arthroplasty, and low-grade infections are particularly difficult to diagnose with use of conventional culture methods. Real-time polymerase chain reaction is a potentially viable way to overcome this detection problem as it is a more rapid and sensitive technique. In the current study, we used intraoperative polymerase chain reaction identification combined with a simple DNA-release method with ultrasonication to diagnose periprosthetic infections during revision surgery. Thirty revision arthroplasty procedures were included in this prospective study. Surgical specimens were obtained intraoperatively, treated with ultrasonication, and then analyzed with real-time polymerase chain reaction. Methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S rRNA gene universal polymerase chain reaction were performed simultaneously to facilitate both specific and broad-range detection. Specimens obtained from the same sites were also analyzed with microbiologic culture and histopathological evaluation. The specific polymerase chain reaction revealed methicillin-resistant Staphylococcus infection in specimens from six of the thirty operations analyzed in the present study, and the 16S rRNA gene universal polymerase chain reaction analysis was positive for specimens from thirteen operations. Conventional cultures revealed six methicillin-resistant Staphylococcus infections, two Staphylococcus aureus infections, one infection with another Staphylococcus species, and two Streptococcus infections. The sensitivity of the polymerase chain reaction method was 0.87 and the specificity was 0.8 when compared with the combined results of microbiologic culture and histopathological evaluation. The ultrasonication method that we developed for accelerated DNA sample preparation as a replacement for conventional extraction made possible the potential intraoperative identification of periprosthetic infection during revision surgery. The simultaneous detection of methicillin-resistant Staphylococcus and broad-range bacterial infections would be invaluable for the informed selection of antibiotics and also for the formulation of the subsequent treatment strategy (a one-stage or two-stage revision) for the patient.

Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.

Improvement of High-throughput Genotype Analysis After Implementation of a Dual-curve Sybr Green I-based Quantification and Normalization Procedure

The ability to rapidly genotype a large number of individuals is the key to any successful marker-assisted plant breeding program. One of the primary bottlenecks in high-throughput screening is the preparation of DNA samples, particularly the quantification and normalization of samples for downstream processing. A rapid and simple Sybr Green I-based quantification procedure that can be performed in a 96-well format is outlined. In this procedure, a dual standard curve method is used to allow better resolution of dilute samples and to reduce fluorescence value variation between samplings. A method to quickly normalize samples, and the importance of normalization, is also explored. We demonstrate that successful fragment amplification of a Theobroma grandiflorum (Willd ex Spreng) Schum. population is increased from 70% to 98% when each DNA extract is quantified and normalized as opposed to quantifying only a subset and normalizing all the samples based on the average of that subset. Improved microsatellite amplification was also observed among individuals in the monocot genus Phaedranassa Herb. ssp. Additionally, when our normalization method is applied to a Persea americana Mill. population, 97% of the samples normalized to 4 ng·μL−1 amplify at least three of six microsatellite regions, whereas only 30% of the samples below 4 ng·μL−1 (i.e., samples that could not be normalized) amplify at least three regions. We describe an undemanding method to quantify and normalize a large number of samples, which can be done manually or can be automated.

No Association betweenMatrix Metalloproteinase-1orMatrix Metalloproteinase-3Polymorphisms and Breast Cancer Susceptibility: A Report from the Shanghai Breast Cancer Study

Matrix metalloproteinase (MMP)-1 (interstitial collagenase) and MMP-3 (stromelysin) are structurally related multifunctional enzymes that are involved in physiologic and pathologic tissue remodeling ([1][1], [2][2]). Known to contribute to breast cancer invasion and metastasis ([3][3]-[5][4]), roles

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.

Toward a Simplified Microfluidic Device for Ultra-Fast Genetic Analysis with Sample-In/Answer-Out Capability: Application to T-Cell Lymphoma Diagnosis

If microfluidic devices capable of rapid genetic analysis are to affect clinical diagnostics, they ultimately must be capable of carrying out more than ultra-rapid electrophoretic separations. The last half decade has seen a groundswell of activity in defining miniaturized DNA sample preparation methodologies that can be integrated with chip-based electrophoretic separations. Successfull integration of PCR-based DNA amplification and solid-phase DNA sets the stage for integrated microminiaturized analytical systems with sample in-answer out capabilities. Here we provide a brief review of the state of the art on the microfluidic integration of sample preparation processes with discussion of several systems with highly integrated capabilities, including one capable of detection of infectious agents present in complex biofluids in less than 30 min. This overview is used as a launch point to discuss the design and functionality of similar devices capable of accepting a whole blood or fine-needle aspirate sample, purifying the DNA, amplifying target sequences of the T-cell receptor-γ gene, and eletrophoretically resolving the products for detection of a signature consistent with monoclonality. We describe the details of the early experimental success in defining the individual chip-based processes required for an integrated T-cell lymphoma chip, with a vision to a device that provide sample in-answer out capabilities for diagnosing certain blood cancers in roughly 1 h.

Bacterial DNA Sample Preparation from Whole Blood Using Surface-Modified Si Pillar Arrays

A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip.

Analysis of DNA sequencing systems based on capillary electrophoresis

A method for characterization and optimization of DNA sequencing systems based on capillary electrophoresis is proposed. A model is developed that relates the read length of a sequencing run and the parameters of DNA sequencing systems such as the properties of the separation medium, capillary length, injection time, concentration of the injected DNA sample, detection sensitivity, system noise, and the illumination power. The optimization of DNA sequencing systems will allow determination of optimum regimes for preparation, injection, and separation of DNA samples needed for achieving a read length required. The method proposed is applied to the characterization of capillary-electrophoretic DNA sequencing systems based on single-photon detection.

Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 102 and 4 × 101 CFU ml−1 were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 101 to 108 CFU ml−1. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.

Genetic profile of the arylamine N-acetyltransferase 2 coding gene among individuals from two different regions of Brazil

Arylamine N-acetyltranferase 2 is the main enzyme responsible for the isoniazid metabolization into hepatotoxic intermediates and the degree of hepatotoxicity severity has been attributed to genetic variability in the NAT2 gene. The main goal of this study was to describe the genetic profile of the NAT2 gene in individuals from two different regions of Brazil: Rio de Janeiro and Goiás States. Therefore, after preparation of DNA samples from 404 individuals, genotyping of the coding region of NAT2 was performed by direct PCR sequencing. Thirteen previously described SNPs were detected in these Brazilian populations, from which seven: 191 G > A; 282 C > T; 341 T > C; 481 C > T; 590 G > A; 803 A > G and 857 G > A are the most frequent in other populations. The presence of so-called ethnic-specific SNPs in our population is in accordance with the Brazilians’ multiple ancestry. Upon allele and genotype analysis, the most frequent NAT2 alleles were respectively NAT2*5B (33%), NAT2*6A (26%) and NAT2*4 (20%) being NAT2*5/*5 the more prevalent genotype (31.7%). These results clearly demonstrate the predominance in the studied Brazilian groups of NAT2 alleles associated with slow over the fast and intermediate acetylator genotypes. Additionally, in Rio de Janeiro, a significantly higher frequency of intermediate acetylation status was found when compared to Goiás (42.5% versus 25%) ( p = 0.05), demonstrating that different regions of a country with a population characterized by a multi-ethnic ancestry may present a large degree of variability in NAT2 allelic frequencies. This finding has implications in the determination of nationwide policies for use of appropriate anti-TB drugs.

Comparison of commercial DNA extraction kits and sample preparation for extraction of bacterial DNA from different fractions of whole rumen fluid

Previous work has shown that the biohydrogenation of fatty acids is different in solid and liquid fractions prepared from rumen fluid (Dorel et al. , 2005). This study aimed to relate this observation to differences in the microbial population in the different fractions. Molecular techniques allow the rapid investigation of microbial numbers and diversity in environmental samples. However, description of the microbial diversity can be affected by the efficiency of DNA extraction from samples as it is known that it is harder to quantitatively extract DNA from some microbes than others (Clement and Kitts, 2000). The aim of this study was to investigate different protocols for preparing samples and extracting DNA from different fractions of rumen fluid, in terms of both the numbers and diversity of bacteria that could be recovered.

CHARACTERISTIC FEATURES OF ISOLATION OF TOTAL DNA FROM PATHOGENIC AND SAPROPHYTE <i>Ascomycetes</i><i>BAsidiomycetes AND deuteromycetes </i>FUNGES

The micromethod of rapid preparation of DNA samples from fungi genus Candida, Aspergillius , Pleurotus и Lentinus have been developed. Showed, that prepared samples of DNA have high quality of clearness and no visual degradation.

Simultaneous cell lysis and bead trapping in a continuous flow microfluidic device

Continuous flow cell lysis by electroporation (EP), and cell and bead trapping by dielectrophoresis (DEP) have been demonstrated on a dielectrophoretic microfluidic chip. The results of lysing human white blood cells (WBC) and murine clonal cells (MN9D) by electroporation showed that lysis rates of up to 80% were achieved at 3 MHz and a flow rate of 30 μl/min. It was also shown that the lysis reactor can function as a dielelectrophoretic filter for trapping silica beads which selectively bind to DNA. The combination of cell lysis, DNA binding to beads, and bead separation assay in a single structure offers a step forward in the development of an efficient and compact system for fully automated microfluidic DNA sample preparation. The continuous flow protocol addresses major challenges faced in sample preparation such as lysate clogging, cell sedimentation, and cell agglomeration.

The use of capillary electrophoresis for DNA polymorphism analysis.

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5 ′ nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations 1 See, e.g., U.S. Patents 5,712,098 and 6,025,130. 1 . This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

High-speed plasmid isolation using 96-well, size-exclusion filter plates.

The accelerating pace of genomics analysis has necessitated the abbreviation of DNA sample preparation protocols. We have developed a size-exclusion-based system for the rapid isolation ofplasmid DNA in a 96-well microplate format. This high-speed protocol employs a modified alkaline lysis methodfor the preparation of the bacterial lysate, followed by three short vacuum filtration steps. Unlike traditional bind/wash/elute methods, there is no need to use chaotropic salts or ethanol. The samples are recovered from the top side of the MultiScreen96 PLASMID plates. Starting with bacterial cell pellets, the entire prycedure for purifying the plasmid DNA can be performed in 30 min with a multichannel pipettor. The high yields, reproducibility, and quality of the plasmids make this system a good choice for any cloning or DNA sequencing operation.

Development of Plastic Microfluidic Devices for Sample Preparation

Microfluidics technology shows excellent potential for applications in biotechnology, chemical sensing, and drug delivery. The use of microfluidics results in cycle time reduction, reagent cost and labor intensity savings due to the benefits of miniaturization, and functional integration. As a result, miniature DNA sample preparation and analytical devices may potentially become part of a point-of-care systems for rapid medical diagnoses. Similar diagnostic devices will benefit veterinary and food safety applications. In this paper, we discuss design, fabrication, and testing of plastic microfluidic devices for on-chip genetic sample preparation. These fabrication methods are being used to produce components of a complete genetic sample preparation micro-system. The detailed discussion on the development of micro-PCR (polymerase chain reaction) devices and bio-channel hybridization arrays is given. We also describe a path to further individual component integration.

A rapid and efficient method for preparation of genomic DNA suitable for analysis of both high and low molecular weight DNA fragmentation during neuronal apoptosis

In this paper, we describe a fast (within 1 h) and efficient method for the preparation of intact genomic DNA suitable for the analysis of DNA integrity in neuronal cells undergoing apoptosis. The procedure involves the embedding of apoptotic cells into low-melting point agarose, in situ cell lysis and purification of DNA samples followed by resolution of fragmented DNA by agarose gel electrophoresis. The method has no restrictions on the number of simultaneous DNA preparations, allows detection of both high and low molecular weight DNA fragments, and is suitable for the preparation of DNA samples from cultured cells, fresh or frozen tissues, as well as from purified nuclei. This procedure is also applicable for the preparation of intact genomic DNA for the megabase-scale analysis. Theme A: Development and regeneration Topic: Neuronal death

PCR-Oligonucleotide Ligation Assay from Dried Blood Spots

PCR followed by oligonucleotide ligation assay (PCR-OLA) is a molecular method that can be used for the detection of nucleotide sequence variants. During the past 10 years it has been used successfully for numerous diagnostic applications (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). PCR-OLA is based on the enzymatic ligation of two oligonucleotides that anneal next to each other onto the PCR-amplified target DNA. Even a single-nucleotide mismatch between the oligonucleotides and the template precludes the ligation. Automated PCR-OLA on a microplate format in combination with ELISA detection technology is a powerful method for testing numerous samples (2). One bottleneck for the effective use of PCR-OLA in population screening programs has been the time-consuming and tedious preparation of DNA samples that is necessary to eliminate PCR inhibitors (16). Dried blood spot (DBS) specimens on filter blotters are widely used for collection, storage, and shipping of blood samples in screening programs for newborns (17), and they have been used for genotypic confirmation of positive screening tests (18). PCR inhibitors can be eliminated from DBS specimens by eluting the spots (19)(20), by fixing the spots to the disk with methanol (21)(22), or by extracting DNA from the disk (23)(24). To increase the throughput of PCR-OLA, we treated DBS specimens with methanol and used the methanol-fixed samples for the PCR-OLA analysis of the major mutation (AGUFin) (25), which is responsible for Finnish-type aspartylglycosaminuria (AGU; McKusick 208400). We tested the methanol treatment on DBS specimens collected on five different types of glass fiber or filter paper blotters. The results show that PCR-OLA from methanol-treated DBSs enables rapid and reliable detection of genetic variances. We …

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil.

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.