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At least 523 records · Page 29Linked to original sources

PubMed2003

Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA.

The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice ('cGMP') and produce milligram-to-kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.

Bioreactors↗

PubMed2005

Optimized sequence retrieval from single bands of temperature gradient gel electrophoresis profiles of the amplified 16S rDNA fragments from an activated sludge system.

Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single bands were excised, and a clone library was constructed for each. Sequence heterogeneity in each single band was found to be significantly overestimated due to single-stranded DNA (ssDNA) contamination formed during the PCR amplification, since only 10-60% of library clones of each single TGGE band had identical migration behavior compared with the parent band. Three methods, digestion with mung bean nuclease, optimization of PCR amplification, and purification via denatured polyacrylamide gel electrophoresis (d-PAGE), were compared for their ability to minimize ssDNA contamination, with the last one being the most efficient. After using d-PAGE to minimize ssDNA to a nearly nondetectable level, 70-100% of library clones for each single TGGE band had identical migration compared with the parent band. Several sequences were found in each of six single bands, and this co-migration could be predicted with the Poland software. The predominant bacteria of the activated sludge were assessed via a combination of sequence retrieval from each single TGGE band and band intensity analysis. Only beta and alpha subclasses of the Proteobacteria were detected, 93.8% and 6.2%, respectively. Our work suggests that prior to constructing a clone library to retrieve the actual sequence diversity of a single DGE band, it is advisable to minimize ssDNA contamination to a nondetectable level.

Alphaproteobacteria↗

PubMed2003

The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

Animals↗

PubMed1992

[Detection of Toxoplasma gondii by "Polymerase Chain Reaction" (PCR) technique in AIDS infected patients using the repetitive sequence TGR1E].

A repetitive original DNA sequence, TGR1E, was cloned and sequenced, then used to develop a polymerase chain reaction (PCR) test for detecting Toxoplasma gondii. Preliminary studies were performed using purified T. gondii DNA or a lysate of purified T. gondii cells [7], with or without a leukocyte lysate. A negative correlation was evidenced between sensitivity of the test and the amount of cellular debris contaminating the DNA to be amplified. Nevertheless, the method was tested on 100 clinical specimens subjected to lysis using the same method. Among the 88 specimens from AIDS patients, four were positive by conventional diagnostic tests and by PCR. Among the 12 specimens tested as part of evaluations for the prevention of congenital toxoplasmosis, PCR failed to detect the positive results yielded by conventional tests on two amniotic fluid specimens. No false positive result was seen with the PCR method.

Acquired Immunodeficiency Syndrome↗

PubMed1999

False human hematopoietic cell lines: cross-contaminations and misinterpretations.

The risk of adventitious contamination and subsequent overgrowth of cell lines by unrelated cells is a potential and often recurring problem where cells are grown and studied. This problem of intraspecies and interspecies cross-contamination among human cell lines has been recognized for over 25 years; incidences of cell cross-contamination between 17 and 35% have been reported. The most useful methods to detect human cell cross-contamination are DNA fingerprinting and cytogenetic analysis, each complementing the other. Using this combination, we found that in total 14.8% of the human hematopoietic cell lines received either from the original investigator (n = 117 cell lines) or from secondary sources (n = 72 cell lines) were cross-contaminated with another hematopoietic cell line and were thus false cell cultures. Another problem relates to the fact that not every cell line established from a patient with a hematopoietic malignancy is a malignant cell line; unintended immortalization of non-malignant B cells by 'passenger' Epstein-Barr virus (EBV) leads to the establishment of B-lymphoblastoid cell lines (termed EBV+ B-LCLs), an event which is much more frequent than the establishment of a 'true' leukemia-lymphoma-myeloma cell line. These EBV+B-LCLs are most often (albeit not always) unrelated to the malignant clone. The misinterpretation of such EBV+ B-LCLs as true malignant hematopoietic cell lines (particularly in research areas investigating B cell-derived neoplasms such as myeloma) and the indiscriminate use of these cell lines may render some of the results of such studies irrelevant to the pathobiology of the disease concerned. However, a combination of markers commonly allows for an accurate determination of the nature of EBV+ B-LCLs: immunoprofile, cellular morphology, EBV status, and karyotype. In summary, the continuous need for vigilant quality and identity control procedures is emphasized by the high incidences of cross-contaminated cell lines. Most laboratories using cells cultured in vitro maintain multiple cell lines. Such cell lines should be monitored regularly for their identity and specific characteristics in order to prevent invalidation of research work due to incidents of cell line cross-contamination or misinterpretation.

Cell Culture Techniques↗

PubMed1990

Risk of tumor induction in vivo by residual cellular DNA: quantitative considerations.

Certain genes, termed oncogenes, have the ability to transmit the transformed phenotype from tumor cells to hitherto normal cells in vitro and in vivo. This observation is the basis for the concern that medicinal products derived from tumor cell lines could contain possibly tumorigenic contaminating cellular DNA. A comparison between the number of oncogenes necessary for the induction of tumors in a suitable animal model and the quantity in 100 pg residual cellular DNA was carried out. It is shown that the latter contains less than a billionth of the number needed to induce tumors in half of the animals tested. Some factors implicit to this calculation such as the validity of the model system, the linearity of the dose response curve, the concept of multistep carcinogenesis, the amplification of oncogenes, and the transfection enhancing effect of other constituents of the drug are discussed. In summary, 100 pg residual cellular DNA per dose seems to be a safe and, therefore, an acceptable upper limit provided that some factors such as excessive amplification of oncogenes, co-purification of oncogenic DNA with the product or some transfection mediating activity can be excluded.

Animals↗

PubMed1983

Localization of DNA polymerase alpha on the nuclear membrane in sea urchin embryos.

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.

Animals↗

PubMed1995

Comparison of methods of DNA extraction from stream sediments.

In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cells are removed from sediments prior to lysis (the Jacobsen method [C. S. Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462, 1992]). DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗

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

Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

Animals↗

PubMed1993

Detection of human papillomavirus DNA and mRNA using synthetic, type-specific oligonucleotide probes.

Type-specific 30'mer-36'mer oligonucleotide probes complementary to mRNA transcribed from the E6 and E7 open reading frames of HPV 6b/11, 16, 18 and 33 were designed using the published nucleotide sequences. As oligonucleotides are easily and relatively cheaply synthesized in large amounts and are free of vector DNA, they were assessed for potential use in routine clinical detection and typing of HPV. Multiple Southern and dot blots of cloned HPV 6b, 11, 16, 18, 31 and 33 DNA, and of DNA extracted from cell lines carrying integrated HPV 16 and 18 genomes were prepared. In addition, Northern and dot blots of RNA extracted from the HPV-containing cell lines HeLa, CaSki and SiHa, were also prepared. All filters were first probed with the oligonucleotide and then with the corresponding full-genomic HPV DNA probe and their relative sensitivities and specificities compared: both probe types were labelled with 32P. The oligonucleotide probes were all as specific as the full-genomic probes for Southern, DNA and RNA dot blot hybridisations. The HPV 16 and 18 oligonucleotide probes detected HPV transcripts of the appropriate sizes in the cell line RNA. For DNA detection, oligonucleotide probes were up to 10 times less sensitive than the full-genomic probes, but for RNA detection, they were more sensitive. The sensitivity for both HPV DNA and RNA detection could be improved by using two type-specific oligonucleotide probes in combination, without reducing the specificity. The ease of preparation and handling of oligonucleotide probes, together with their lack of contaminating vector DNA, suggests that they may have some advantages over full-genomic probes for the clinical detection and typing of HPV.

Base Sequence↗

PubMed2004

Two novel real-time reverse transcriptase PCR assays for rapid detection of bacterial contamination in platelet concentrates.

The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services.

Bacteria↗

PubMed1979

Changes in mitochondrial DNA in cardiac hypertrophy in the rat.

We studied DNA (mtDNA) replication in adult female rat hearts undergoing hypertrophy secondary to constriction of the ascending aorta. MtDNA was measured in isolated mitochondria by a fluorometric method adapted for that purpose. The conditions for removal of contaminating nuclear DNA were developed, and the purity of the mtDNA was assessed from its molecular conformation (open and closed circles) and by renaturation-kinetic analysis. The mtDNA concentration in mitochondria, expressed as micrograms of DNA per milligram of mitochondrial protein, increased 2, 4, and 7 days postoperatively by 21, 73, and 98%, respectively. Similar results were obtained when mtDNA was expressed per nonomole of cytochrome a. The population of replicative intermediates of mtDNA was analyzed by electron microscopy. In normal hearts, we observed molecular forms characteristic of animal mtDNA, such as circular monomers and dimers, catenated molecules, D-loops, expanded D-loops, and gapped molecules. D-loop frequency, which was near 50% in the mtDNA of control hearts, was markedly reduced to 5-7% in hypertrophying hearts. This result indicates that the increase in replicative flux of mtDNA is associated with the removal of a block in the conversion of D-loops to other intermediates.

Animals↗

PubMed2000

DNase I induced DNA degradation is inhibited by neomycin.

Preparations of antimicrobials from biotechnological sources containing nucleic acids may serve as vector for the dissemination of resistance genes. An essential prerequisite for the acquisition of a new resistance phenotype in a transformational scenario is the availability of physically intact DNA molecules capable of transforming competent microorganisms. DNA is thought to be an easy target for catabolic processes when present in the natural habitat of bacteria (e.g. gastrointestinal tract, soil) due to the overall presence of nucleolytic enzymes. Aminoglycoside antibiotics are known to display a strong affinity to nucleic acids rendering these compounds to be primary candidates for exerting DNA protective functions in the gastrointestinal tract when applied orally during antibiotic chemotherapy. Using a DNase I protection assay it could be demonstrated that neomycin B at a concentration of 2 mM completely inhibited degradation of plasmid DNA in vitro. No inhibition of degradation was observed with streptomycin and kanamycin and the non-aminoglycoside antibiotics oxytetracycline and ampicillin under identical assay conditions. Thus, neomycin preparations may be able to promote structural integrity of contaminating DNA-fragments in DNase-rich environments.

Ampicillin↗

PubMed2003

Filtration based DNA preparation for sexual assault cases.

Police departments in the United States currently have as many as 500,000 unprocessed swabs taken from rape victims. The standard method for purifying sperm from these swabs is to resuspend first all cells and to digest selectively the excess of the victim's epithelial cells. The intact sperm are then separated from the contaminating solubilized DNA by centrifugation, careful removal of supernatant, and extensive washing of the sperm pellet, all steps that are difficult to automate. Vacuum driven filtration is an alternative method for separating sperm from digested epithelial cells that requires only pipetting steps and can be readily automated in a 96 well format. Sperm DNA is enriched 45-fold using this process and the yield of PCR ready DNA is roughly 20% of the amount originally present on the swab.

Cell Separation↗

PubMed1997

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.

Animals↗

PubMed1988

Validation of the use of 32P-orthophosphate as a probe for monitoring DNA replication in pancreatic islet cells.

This study aims at validating the use of 32P-orthophosphate as a probe for monitoring DNA replication instead of [3H]-thymidine where the specific activity of the latter is affected by perturbation of the cellular environment. It was found that to obtain a clean DNA fraction for determining the incorporation of the label, protein must be removed by the use of Proteinase K, and RNA by alkaline hydrolysis. Alkaline hydrolysis of RNA was found superior to RNAse digestion in not leaving behind large RNA fragments that may contaminate the DNA fraction.

Animals↗

PubMed1994

[Fast detection of Alu DNA in human tissues by Alu-PCR].

This paper reports a direct PCR method for the detection of Alu DNA in 18 different kinds of human tissue using primers of Alu 9.1 and Alu 9.2 without DNA extraction. The results showed that besides fresh tissues, some formalin fixed paraffin embedded tissues can be correctly analyzed with PCR. Different amounts of PCR products were obtained from different types of tissues. The duration of fixation in formalin is an important factor exerting an influence on the quantity of the high molecular weight DNA and the results of PCR. No obvious influence of the duration of storage of paraffin embedded tissues on the PCR results was observed. This method is quite simple, sensitive and rapid to perform. It can be performed under a condition of avoiding contamination of DNA and DNase as well as the detrimental phenol and chloroform.

Base Sequence↗