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At least 613 records · Page 34Linked to original sources
PubMed2005

Early diagnosis and monitoring of superficial transitional cell carcinoma by microsatellite analysis on urine sediment.

It has recently been shown that allelic abnormalities, detected by microsatellite analysis of the DNA extracted from urine sediment, can be successfully used for the detection of transitional cell carcinoma (TCC) of the bladder. The diagnostic accuracy of urinary cytology, urinary bladder cancer (UBC) marker, bladder tumor antigen (BTA) and microsatellite sequence alterations was compared in 42 patients who were recruited for the study. Of them, 30 had been diagnosed with TCC at cystoscopy plus biopsy (group A). Seven patients without any apparent lesions after trans-urethral resection (TUR) and 6 subsequent weeks of endovesical administration of bacillus Calmette-Guerin (BCG), had irritative symptoms. None of them had positive cytology or TCC bladder mucosa biopsies (group B). In the control group were 5 other subjects who were affected by benign prostatic hypertrophy and candidates for prostatectomy (group C). Urine and blood samples were obtained from all of the patients before surgery. Tumor tissue and normal mucosa samples were taken from groups A and C during surgery. Different urinary sediment analyses were performed by using both nuclear medicine and molecular tests. UBC and BTA-t analyses were carried out using monoclonal antibody tests while microsatellite analyses were performed using extracted DNA and electrophoresis of polymerase chain reaction (PCR) products on 13 different primers. Urinary cytological examinations were carried out using the Autocyte Preparation System(R). Urinary cytology confirmed the presence of TCC in 13.3% of patients. The BTA-t marker allowed the identification of 73.3% of cancers with 50% specificity; the UBC marker identified 63.3% of the cases with 41.6% specificity. Microsatellite analysis permitted the identification of 83.3% of the tumors with 100% specificity. DNA analysis demonstrated high sensitivity in patients affected by superficial (81.4%) or G1 (80%) tumors, even when cytological studies demonstrated little or no sensitivity. Microsatellite analysis is a highly-sensitive and specific marker for TCC diagnosis and its monitoring, especially in patients with low-stage and low-grade tumors. Other testing procedures failed to increase urinary cytological diagnostic significance.

Antigens, Neoplasm↗
PubMed2005

DNA assays for detection, identification, and individualization of select agent microorganisms.

The purpose of this article is to review the status of DNA assays used for the detection, identification, and individualization of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, and Brucella abortus. These select agent microorganisms are historically significant as they have either been used or experimented with as a bioweapon or as a terrorist agent and are the subject of intense research in the areas of biodefense and bioforensics. If the presence of a biological agent is suspected, sensitive and specific assays for rapid detection and identification are necessary. However, DNA methods for identification of the sample may also be applied in order to individualize the strain and potentially determine the source of the microorganism. Methods used at the Armed Forces Institute of Pathology (AFIP) for select agent microbial DNA analyses include DNA extraction, DNA quantitation, real-time polymerase chain reaction (real-time PCR) of genetic targets unique to the select agent microorganism, microbial 16S ribosomal RNA gene DNA sequencing, amplified fragment length polymorphism polymerase chain reaction (AFLP-PCR), and more recently, repetitive element polymerase chain reaction (REP-PCR) DNA fingerprinting. The methodologies of 16S ribosomal RNA gene DNA sequencing and DNA fingerprinting of microorganisms are well established within the field of diagnostic microbiology for DNA identification purposes, as well as DNA typing for epidemiological and genetic relatedness studies. 16S ribosomal RNA gene DNA sequencing and AFLP DNA fingerprinting have been validated at the Armed Force Institute of Pathology (AFIP) laboratory for identification purposes and can be used as a possible strain typing tool for Bacillus anthracis, Yersinia pestis, Francisella tularensis, as well as Brucella and Burkholderia species. The continued development and implementation of new DNA based methods with increased sensitivity and defined specificity will be particularly useful for the detection of residual microbial DNA signature in situations where the microorganism has been rendered nonviable by decontamination procedures or not able to be cultured on microbiological media.

Biological Warfare↗
PubMed1982

Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA. Purification by chromatography on malachite green DNA affinity resin.

Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA (1.715 satellite chromatin) were purified in order to test for differences between satellite chromatin and unfractionated chromatin. The final purification procedure involved digestion of isolated nuclei with Eco RI restriction endonuclease, solubilization of a fraction of the chromatin by lysis of the nuclei at low ionic strength, and chromatography of the soluble chromatin fragments on malachite green DNA affinity resin. Digestion of chromatin within steer kidney and thymus nuclei by Eco RI reached limits at which 22% and 37%, respectively, of the potential sites for the enzyme in the 1.715 satellite chromatin were cleaved. A test of the 1.715 g ml-1 satellite DNA extracted from digested thymus nuclei showed it was not significantly nicked at Eco RI sites. Fractionation of soluble chromatin fragments produced by Eco RI digestion on the basis of size using either sucrose gradient centrifugation or chromatin gel electrophoresis yielded 1.715 satellite chromatin of 60-80% purity. However, chromatography of the soluble chromatin fragments on columns of malachite green resin gave 1.715 satellite chromatin of higher purity and in greater amounts than could be obtained with the sucrose gradients or chromatin gels. Using malachite green resin, hundreds of micrograms of 1;715 satellite chromatin could be obtained with a purity that was usually at least 95% as determined by melting of the extracted DNA.

Animals↗
PubMedDate not supplied

Polymerase chain reaction amplification of Guthrie card deoxyribonucleic acid: extraction of nucleic acid from filter matrices.

This study evaluated two deoxyribonucleic acid (DNA) extraction methods for Guthrie card bloodspots. Our water-based extraction technique was compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to extract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chain reaction (PCR) for a 491 bp region encoding the cystic fibrosis delta F508 mutation was performed and PCR products electrophoresed on polyacrylamide gels and stained with ethidium bromide. Amplification of GFX-extracted DNA required low sample volume (1 to 5 microL) indicating the presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 punch) were comparable to our standard conditions and represented 0.16-0.80 microL whole blood (20 to 40 ng DNA). The GFX-extracted DNA was found to be stable (6 mo, -15 degrees C). Performance time for the GFX method was less than our standard water-based extraction (about 30 min), but more costly. The GFX extraction kit was adaptable for limited sample (i.e., extraction of a 3 mm punch yields 20 amplifications). The GFX extraction kit provides a useful means to standardize Guthrie card DNA extraction.

Blood Chemical Analysis↗
PubMed1998

Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption.

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (10(8) and 10(7) CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10(8) CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P < or = 0.005), 10(7) CFU of C. neoformans (P < or = 0.05), and 10(7) CFU of A. fumigatus (P < or = 0.01). Yields were within the same range for 10(8) CFU of C. neoformans and l0(7) CFU of C. albicans for both HSCD extraction and PC extraction. For 10(7) CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P < or = 0.05). Yields obtained from 10(8) and 10(7) CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

Chloroform↗
PubMed2005

Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

Animals↗
PubMed1990

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. II. Extraction of spooled DNA and its application to Southern blot hybridization analysis.

In our previous report, we described a new fixation and paraffin-embedding method (the AMeX method) that preserves many of the antigens that are normally destroyed by routine formalin fixation. The current study was conducted to examine the preservation of high-molecular-weight DNA in tissues processed by this method. DNA was extracted from AMeX-processed tissue sections after deparaffinization by the same method as that used to extract DNA from fresh tissues. The total amounts of DNA extracted from 10 mg each in wet weight of AMeX-processed and fresh mouse liver tissues were identical. In tissues of malignant lymphoma, the total amount of spooled DNA extracted from 50 sections, each 20 microns thick, was about 8 micrograms/mm2. The electrophoretic pattern of DNA digested with restriction endonucleases on agarose gel from AMeX-processed tissue sections did not differ from that of fresh materials. Southern blot hybridization analysis also revealed that the mobility of specific DNA fragments was identical for AMeX-processed and fresh tissues. The AMeX method was thus proved to be a versatile multipurpose tissue-processing procedure, which is expected to provide important information regarding the correlation between morphology, phenotypic expression, and gene alteration.

Blotting, Southern↗
PubMed2005

Evaluation of three methods for effective extraction of DNA from human hair.

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.

Cheek↗
PubMed1993

Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures.

Sonication procedures for the extraction of DNA from forensic-type semen specimens have been developed, which, when compared to currently utilized sperm DNA extraction techniques, are simple, rapid and result in comparable DNA yields. Sperm DNA extraction by sonication was performed on whole semen, seminal stains, buccal swabs and post-coital specimens. Ultrasound disruption of sperm cells and their ultimate release of cellular DNA has been conducted in the presence of sperm wash buffers followed by organic extraction or Chelex 100 with little or no compromise to DNA quality, quantity or amplifiability. Two advantages of sonication over currently used forensic techniques to extract sperm DNA include 1) sperm DNA extraction that occurs within five minutes of sonication compared with an hour or greater for water bath incubations in classic enzyme digestion DNA extractions and 2) one less preparatory step with the Chelex/sonication protocol and three less steps with the sonication/organic protocol compared with other procedures thus eliminating potential sample-to-sample cross-contamination. Sperm DNA extracted by optimum sonication procedures was used for forensic HLA DQ alpha typing and restriction fragment length polymorphisms analysis without any adverse effects on typing results.

Cheek↗
PubMed1999

Extraction of DNA from decomposed human tissue. An evaluation of five extraction methods for short tandem repeat typing.

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed biological material, a fact that has to be taken into account when choosing the appropriate casework methods. In this paper we report the evaluation of five different DNA extraction methods, namely the phenol-chloroform, the silica based, the InstaGene Matrix (BioTest), the glass fiber filter, and the Chelex based methods. The substrates for the analyses are decomposed human liver tissue specimens from forensic autopsy cases. Extracted DNA was quantified and DNA profiled by a set of seven STRs. We have compared laboratory time consumption and costs of the five methods, showing that the Chelex method is the more rapid and less expensive of the methods, the phenol-chlorophorm and silica extractions being the most time consuming and resource demanding ones. A full profile was obtained by the silica method in nine out of ten cases and this method failed to give a reliable type in four out of 70 STR analyses. The phenol-chlorophorm and the glass fiber filter methods failed in 16 analyses, the InstaGene Matrix (BioTest) in 25 and the Chelex extracts in 56 of the 70 STR analyses. By multiple logistic regression we show that the difference between the silica procedure and the other methods are statistically significant. In our hands, the silica gel extraction procedure is an obvious choice when the biological material available is decomposed human tissue--even if this procedure is one of the more laborious ones.

Autolysis↗
PubMed1990

Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors.

The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity. These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase. This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J. Laffan and W. Firshein, J. Bacteriol. 169:2819-2827, 1987). Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation. Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives. Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase. Some regulatory mechanisms of the kinase system may also be retained by the complex. Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex.

Bacillus subtilis↗
PubMed2001

Extraction and detection of baculoviral DNA from lake water, detritus and forest litter.

AIMS: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. METHODS AND RESULTS: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt-/lacZ+. The detection limit of PCR amplification was routinely about 4.1 x 102 occlusion bodies (OBs) 450 microl-1 lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 103 and 2.08 x 104 OBs 0.5 g-1 dry weight, respectively. CONCLUSION: The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.

Baculoviridae↗
PubMed2004

LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts.

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis, the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45 degrees C treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

Animals↗
PubMed1991

Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesis.

Extracts from HeLa cells were used to study the susceptibility of repair synthesis in UV-irradiated plasmid DNA to inhibition by exogenously added nucleic acid. Purified DNA restriction fragments have little inhibitory effect on repair synthesis. However, activated calf thymus DNA fragments, genomic DNA fragments in cell extracts, and sonicated plasmid DNA all inhibited repair synthesis. Degraded DNA fragments arising from E. coli during bacterial plasmid purification were found to be particularly inhibitory. tRNA is not a potent inhibitor of in vitro repair synthesis. In order to observe efficient DNA repair synthesis mediated by human cell extracts, it is essential to prepare highly purified closed circular plasmid DNA, and we describe a reliable method for doing so.

Cell Extracts↗
PubMed2004

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples.

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).

Animals↗
PubMed1999

Fast and reliable extraction of protozoan parasite DNA from fecal specimens.

BACKGROUND: Polymerase chain reaction (PCR) detection of intestinal protozoa in fecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this study we describe a novel method for DNA extraction from such specimens containing spores and oocysts of Enterocytozoon bieneusi and Cryptosporidium parvum, respectively. METHODS AND RESULTS: Extraction was done using commercial kits modified to maximize the recovery and purity of extracted DNA. In comparison with a procedure we previously reported, we estimate that this method may increase the sensitivity of parasite DNA detection in fecal specimens up to tenfold. An additional advantage of this method is that up to 12 samples may be processed simultaneously within 2 hours. CONCLUSIONS: By using this method, we were able to increase reproducibility of PCR amplification on fecal specimens and significantly reduce the hands-on time required to process the samples.

Animals↗
PubMed2006

Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR.

Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.

Animals↗
PubMed2005

Use of magnetic beads to extract fungal DNA.

Authors compare two methods of extracting DNA from different fungi: the classic method with phenol/chloroform (P/C) and that with magnetic beads. Both were tested on Candida albicans and Cryptococcus neoformans var. neoformans, belonging to the yeast group and Microsporum canis, M. gypseum, Trichophyton rubrum, T. interdigitale, T. ajelloi, Epidermophyton floccosum, belonging to the dermatophytes group. Extraction products underwent polymerase chain reaction (PCR) fingerprinting with the appropriate primers to point out any disagreement in the genomic profiles. After having determined that the genomic profiles obtained from the DNA extracted from the same strain with the two methods correspond perfectly, the authors concluded that the extraction method with magnetic beads from fungal cells is simpler and quicker than with P/C extraction, greatly facilitating the obtainment of fungal DNA.

Arthrodermataceae↗