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PubMed2000

Phylogeny and host specificity of Psoroptic mange mites (Acarina: Psoroptidae) as indicated by ITS sequence data.

We used molecular phylogenetic techniques to study the systematic relationships and host specificity of Psoroptes mange mites, which are pests of numerous domestic and wild ungulates. Phylogenetic analysis of DNA sequence data from the internal transcribed spacer region 1 (ITS1) of nuclear ribosomal DNA indicated that populations of Psoroptes are not host specific. Furthermore, the currently used taxonomy of Psoroptes is not concordant with the phylogeny derived from ITS1. During the course of the study, we discovered apparent paralogous ITS sequences within individual mites as a result of varying polymerase chain reaction reaction conditions. This finding concords with other studies of ITS and suggests a cautious approach when interpreting data from ITS sequences. Host DNA contamination was also found to be a significant problem in data collection, and we report on the development of methods to overcome the problems of contamination in parasitic mites.

Animals↗

PubMed1990

Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA.

The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.

Base Sequence↗

PubMed1993

Detection of low level of human papilloma virus type 16 DNA sequences in cancer cell lines derived from two well-differentiated nasopharyngeal cancers.

Human papilloma virus type 16 (HPV16) related sequences were detected in two EBV-negative nasopharyngeal carcinoma (NPC) cell lines derived from two well-differentiated NPC by polymerase chain reaction. E2 and E6 related sequences of HPV16 were demonstrated using two pairs of primers derived from these two regions. DNA sequence analysis of amplified products excluded the possibility of laboratory viral DNA contamination.

Antigens, Viral↗

PubMed2001

The transforming activity of tumour cell DNA.

Transfection of DNA isolated from a subset of human tumour cells was shown in the early 1980s to be capable of inducing transformation in NIH3T3 cells. This review summarizes the approaches and results of the efforts to measure the transforming activity of genomic DNA and discusses the results in the light of current concerns about the potential hazard associated with DNA contamination of biological material produced by cultured transformed cells.

3T3 Cells↗

PubMed2006

The importance of careful blood processing in isolation of cell-free DNA.

In healthy individuals, the source of cell-free plasma DNA is predominantly apoptotic, whereas, increased plasma DNA integrity is seen in cancer patients. Therefore, it is important to carefully isolate absolutely "cell-free" plasma DNA. Plasma DNA from 30 healthy females was analyzed using 4 PCR amplicons of increasing size, comparing standard blood processing with additional centrifugation steps prior to DNA extraction. Cellular DNA contamination, indicated by positive amplicons >300 bp was eliminated only after the extra centrifugation step. This highlights the importance of careful processing in preparation of cell-free plasma DNA as a tool for cancer detection and we recommend the use of a microcentrifuge spin, prior to DNA extraction.

Blood Chemical Analysis↗

PubMed1992

Positive immunoglobulin gene rearrangement study by the polymerase chain reaction in a colonic adenocarcinoma.

The detection of clonal rearrangements of the immunoglobulin heavy chain gene by the polymerase chain reaction provides a rapid method to differentiate monoclonal from polyclonal B-lymphocyte proliferations. It has been shown to be highly specific and so far, no false-positive results have been described. A case of a poorly differentiated colonic adenocarcinoma that showed a "false positive" clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction technique is reported. DNA contamination was unlikely because of the strict adherence to the laboratory polymerase chain reaction protocol and also the repeated demonstration of the same amplified band in a separate experiment using DNA extracted from another piece of tumor tissue. The apparent monoclonal immunoglobulin heavy chain gene rearrangement in the first polymerase chain reaction may be related to a combination of the paucity of lymphoid cells in the tissue sample and the presence within this small number of lymphocytes of a clonal reactive cell population. It is, therefore, important to correlate the routine microscopic and immunohistochemical findings in the interpretation of polymerase chain reaction results, especially when working with nonlymphoid tumors and lymphocyte-poor lesions.

Adenocarcinoma↗

PubMed1984

Repetitive DNA of Candida albicans: nuclear and mitochondrial components.

We report the isolation and analysis of the rapidly reassociating DNA of the pathogenic, dimorphic fungus Candida albicans. Minicot analysis of whole-cell repetitive DNA suggested that a significant portion of this component was mitochondrial DNA. Genomic blot hybridizations in which radioactive whole-cell repetitive DNA was used as a probe revealed eight major EcoRI bands in the molecular weight range resolved by the gel system used. Isolation and analysis of high-purity mitochondrial DNA have shown that five of these bands are of mitochondrial origin. The remaining three bands are of nuclear origin and represent repetitive sequences that are found in the nuclear genome. Attempts to isolate nuclear DNA that was completely free of mitochondrial DNA contamination were unsuccessful.

Candida albicans↗

PubMed1997

The role of immunocytochemistry and linkage analysis in the prenatal diagnosis of merosin-deficient congenital muscular dystrophy.

Complete or partial deficiency of the laminin alpha2 chain of merosin has been demonstrated in a proportion of children with classical congenital muscular dystrophy and linkage to the laminin alpha2 chain gene (LAMA2) on chromosome 6q2 has been established. As the laminin alpha2 chain is also expressed in the trophoblast, its detection and linkage analysis are useful tools for prenatal diagnosis. We report our experience of seven prenatal diagnoses in families with partial deficiency or total absence of the laminin alpha2 chain in the muscle of the propositi. In five instances, expression of the laminin alpha2 chain in the trophoblast was normal and linkage data suggested that the fetuses were unaffected. In one family, the immunocytochemical studies of the trophoblast showed the absence of laminin alpha2, suggesting that the fetus was affected. Linkage analysis confirmed that the fetus had inherited the two at-risk haplotypes. In one family with partial laminin alpha2 chain deficiency, the haplotype analysis was hampered by maternal DNA contamination. Immunocytochemical analysis of chorionic villus sampling showed a reduction in laminin alpha2 expression. The pregnancy was presumed to be at high-risk and terminated. However, subsequent analysis of fetal DNA indicated that the fetus was probably heterozygous. Our data suggest that immunocytochemical analysis of the trophoblast can detect abnormalities in affected fetuses and gives normal results in unaffected and carrier fetuses. Nevertheless, we recommend that linkage analysis to the LAMA2 locus is also studied in all cases.

Female↗

PubMed2006

qPrimerDepot: a primer database for quantitative real time PCR.

Gene expression studies employing high throughput real time PCR methods require finding uniform conditions for optimal amplification of multiple targets, often a daunting task. We developed a primer database, qPrimerDepot, which provides optimized primers for all human and mouse RefSeq genes. These primers are designed to amplify desired templates under unified annealing temperature. For most intron-bearing genes, primers flank one of the largest introns thus minimizing background noise due to genomic DNA contamination. The qPrimerDepot database can be accessed at http://primerdepot.nci.nih.gov/ and http://mouseprimerdepot.nci.nih.gov/.

Animals↗

PubMed1997

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

Artifacts↗

PubMedDate not supplied

Optimised helper virus-free production of high-quality adeno-associated virus vectors.

BACKGROUND: Clinical development of adeno-associated virus (AAV) requires standardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several strategies have been reported for the approach to the manufacturing problem. We report a helper virus-free process that produces high quality rAAV stocks. METHODS: rAAV were produced by triple transfection, a helper virus-free process. After lysis of the cells in the presence of nuclease, the rAAV produced were purified by HPLC through two ion-exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological activity and for the presence of residual contaminants. The titer of infectious particles and of rep + particles was determined by dRA assay. Contaminating DNA and RNA were determined by fluorescent dye binding and real-time PCR. The protein content of the rAAV stocks was characterised by SDS-PAGE, ELISA test, Western blot and specific enzymatic assays for putative residual contaminating protein. The in vivo biological activity of the stocks was evaluated in mouse muscle. RESULTS: rAAV stocks obtained following this procedure elicit: 2-5 x 10(12) pp/ml; 3-6 x 10(10) ip/ml; < 10(3) rep + particles/ml; <0.3 mUeq/ml of residual benzonase activity; non-detectable Ad or beta-galactosidase proteins; <35 pg/ml of cellular genomic DNA; in vivo expression in mouse muscle without any immune reaction detected. CONCLUSIONS: This work demonstrates the possibility of producing purified high-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors.

Animals↗

PubMed2005

Immobilization of deoxyribonuclease via epoxy groups of methacrylate monoliths. Use of deoxyribonuclease bioreactor in reverse transcription-polymerase chain reaction.

A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis-Menten constant, Km(app), and turnover number, k3app, for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l(-1) and 16 dA260nm min(-1) mg(-1) of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 degrees C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.

Base Sequence↗

PubMed2002

High-sensitivity hybridization assay for quantitation of residual E. coli DNA.

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.

Antibodies↗

PubMed1993

[Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal].

It has been confirmed that water-soluble eumelanins often extracted together with DNAs from natural black hairs act as an inhibitor of Taq DNA polymerase in the polymerase chain reaction (PCR). In the present investigation, an attempt to amplify the non-coding 333-bp region of mitochondrial DNA (mt333DNA) produced the following results: 1) Water-soluble preparations made from chemically synthesized melanin (Sigma products), as well as natural black eumelanins, inhibited the PCR amplification of mt333DNA at concentrations of more than 2 micrograms/ml. 2) Quantitative measurement of Taq DNA polymerase-catalyzed DNA synthesis in terms of the amount of [alpha-32P] dCMP incorporated into activated calf thymus DNA showed that both of the water-soluble melanins had the same inhibition activity as represented by the sigmoidal curve derived from a quadratic equation of melanin concentration. This observation suggested that Taq DNA polymerase combined with two molecules of melanin to form an inactivated complex. 3) Melanins did not appear to affect either the thermostability of Taq DNA polymerase at 94 degrees C, or the step of primer-annealing to template DNAs. On the other hand, we established a simple and useful method for removal of water-soluble eumelanins contaminating DNA preparations from hairs. The method was based on the adsorption of melanins to Bio-Gel. When a Bio-Gel P-60 minicolumn was equilibrated with 10 mM sodium acetate buffer, pH 4.2, water-soluble melanins were completely adsorpted to it whereas DNAs passed through, although the melanins showed incomplete adsorption to the gel when it was equilibrated with TE (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Mitochondrial↗

PubMed1997

Techniques for application of faecal DNA methods to field studies of Ursids.

We describe methods for the preservation, extraction and amplification of DNA from faeces that facilitate field applications of faecal DNA technology. Mitochondrial, protein encoding and microsatellite nuclear DNA extracted and amplified from faeces of Malayan sun bears and North American black bears is shown to be identical to that extracted and amplified from the same individual's tissue or blood. A simple drying agent, silica beads, is shown to be a particularly effective preservative, allowing easy and safe transport of samples from the field. Methods are also developed to eliminate the risk of faecal DNA contamination from hair present in faeces.

Animals↗

PubMed2001

Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.

PURPOSE: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. METHODS: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis delta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. RESULTS: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. CONCLUSIONS: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.

Cystic Fibrosis↗

PubMed1998

DNA template amplification genetic studies on archival human preimplantation biopsied microtubed cell samples.

Biopsied cell samples can remain in storage following an initial, unequivocally successful preimplantation genetic diagnosis (PGD). The fidelity of the DNA template for polymerase chain reaction (PCR) amplification of microtubed human preimplantation embryonic cells stored at -70 degrees C for extended periods of time (6 months to 4 years) has been assessed. PCR protocols and specific nested primer sets for the beta-globin, ZP3 and CA repeat motif successfully used for previous human embryo PGD research were employed for these studies. The results show that the DNA template of microtubed biopsied blastomere and mural trophectoderm cell samples stored for periods of up to 4 years may be successfully amplified by PCR. Specific gene sequences were able to be analysed at the 1-2 or 3-5 biopsied cell level with a 71-100% success rate. Analysis of DNA fragments amplified from the CA dinucleotide repeat locus showed that in 8/9 samples both alleles were amplified at the cellular level. No DNA contamination was detected in the stored microtubed samples, or in the experimental controls.

Actins↗

PubMed2002

Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR.

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.

Bacteria↗