PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA contamination”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed2001

Efficient isolation of total RNA from Clostridium without DNA contamination.

Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.

Blotting, Northern↗
PubMed2005

Resistance of degraded hair shafts to contaminant DNA.

We have investigated the susceptibility of degraded human hair shaft samples to contamination by exogenous sources of DNA, including blood, saliva, skin cells, and purified DNA. The results indicate that on the whole hair shafts are either largely resistant to penetration by contaminant DNA, or extremely easy to successfully decontaminate. This pertains to samples that are both morphologically and biochemically degraded. We suggest that this resistance to the incorporation of contaminant DNA relates to the hydrophobic and impermeable nature of the keratin structures forming the hair shaft. Therefore, hair samples represent an important and underestimated source of DNA in both forensic and ancient DNA studies.

Blood↗
PubMedDate not supplied

Sensor-based system for rapid and sensitive measurement of contaminating DNA and other analytes in biopharmaceutical development and manufacturing.

The Threshold System provides for rapid and highly sensitive measurements of a variety of analytes at low concentration. The operating principles of the measurement system, including signal detection utilizing a proprietary silicon sensor, are described. The first application to run on the Threshold System is a quantitative assay for picogram levels of total DNA. This assay is of great value for monitoring levels of contaminating DNA in purified and in-process biopharmaceuticals. After sample pretreatment, the total assay time is less than two hours. The assay performance characteristics are described, along with data demonstrating sensitivity, precision, and accuracy. Pretreatment procedures for biopharmaceutical proteins are discussed. Two additional applications on Threshold are introduced. First, a universal ligand-binding assay system provides quantitative measurement of antigens or antibodies, in a variety of biological media, with speed, sensitivity, and precision. Second, preliminary results of rapid DNA-probe measurements on Threshold are presented.

Biosensing Techniques↗
PubMedDate not supplied

[DNA contamination by examination instruments].

Using DNA-analysis systems in forensic examination, conditions without any (iatrogenic) contamination are necessary for analysis of evidence. Failing these conditions a contamination is possible and interpretation of the results may be difficult. Our experiments demonstrated a relevant transfer of DNA-material if contaminated instruments were used.

Autopsy↗
PubMed2001

Pro-inflammatory activity of contaminating DNA in hyaluronic acid preparations.

Hyaluronic acid (HA), an abundant non-sulfated glycosaminoglycan component of the extracellular matrix, has applications in drug delivery, tissue engineering and as an ingredient in cosmetics. HA preparations containing high-molecular-weight polymers are also used in the treatment of inflammatory disorders such as arthritis and interstitial cystitis. Low-molecular-weight fragments derived from HA have been reported to induce pro-inflammatory cytokines such as IL-12 and TNF-alpha, and could therefore potentially exacerbate existing inflammation. We therefore examined the pro-inflammatory activity of HA preparations, since inflammatory reactions are known to occur following administration of HA. We tested low-molecular-weight fragments obtained from seven different HA preparations, either by sonication (approximately equals 3 x 10(5) Da) or by hyaluronidase digestion (approximately equals 1 x 10(4) Da), for the ability to induce the synthesis of IL-12 and TNF-alpha by human monocytic cells. We found that two of the seven HA preparations tested stimulated the synthesis of IL-12 and TNF-alpha by human monocytic cells. We unexpectedly found that the induction of IL-12 and TNF-alpha by these HA preparations was not due to their degradation to low-molecular-weight fragments, since their native high-molecular-weight forms possessed the same ability to stimulate IL-12 and TNF-alpha synthesis, but was due to the presence of contaminating DNA. Treatment of these two HA preparations with deoxyribonuclease I abrogated or reduced the induction of IL-12 and TNF-alpha. It is clear from this study that HA preparations can induce the synthesis of pro-inflammatory cytokines by monocytes. The ability of HA to act as a pro-inflammatory mediator may not, however, be related to the presence of low-molecular-weight HA fragments, but to the presence of DNA. The presence of pro-inflammatory DNA in HA preparations should be evaluated before its use, not only for the treatment of patients with inflammatory disorders, but also before many other applications.

Adjuvants, Immunologic↗
PubMed1991

Use of sulfonation method to determine DNA contamination of cell cultured monoclonal antibodies produced for human therapeutics.

In the past few years, culture of transformed mammalian cells has been widely used to produce natural or recombinant molecules, as monoclonal antibodies (MAb) and cytokines. For therapeutic use, the cellular DNA level must be determined. A number of techniques have been developed to measure the DNA content, based on sequential extraction blotting and hybridization with a labeled DNA probe. The sulphonate marker has been recently introduced by PBS-Orgenics; it allows the determination of picograms (pg) quantities of purified DNA. However, it is not simple to measure in complex biological samples especially when a large amount of protein is present. In considering the following points: 1. Precautions in handling the samples at different steps of preparation; 2. Modifications of the original technique 3. Concentration of samples expected at very low level, we are able to dose up to 2 pg of contaminant DNA per mg of MAb with a satisfactory reproducibility and reliability. This level is required not only to qualify final MAb but also to evaluate the efficiency of the purification process. Efforts are being made to achieve a better sensitivity.

Antibodies, Monoclonal↗
PubMed2001

Quantification of Escherichia coli genomic DNA contamination in recombinant protein preparations by polymerase chain reaction and affinity-based collection.

This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg x mL(-1) of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg x mL(-1) and1 ng x mL(-1) genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.

Base Sequence↗
PubMed2002

Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.

Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

DNA, Bacterial↗
PubMed2000

Human DNA contamination of mortuaries: does it matter?

With the continuing development of extremely sensitive, automated systems for the detection of human DNA from a number of cellular sources, the problem of sample contamination from scenes of crime, cadavers, and the mortuary environment has become a potentially serious issue, with implications for all involved in forensic investigations. A recent survey of 20 mortuaries identified quantifiable human DNA on mortuary work surfaces and instruments which, when amplified, produced in some cases three or more profiles from single site samples. Possible sources of DNA contamination in the mortuary are discussed, along with implications related to its presence and its avoidance during the sampling process. These observations may not be confined to forensic practice.

DNA↗
PubMed1994

Removal of DNA contaminants from therapeutic protein preparations.

Mammalian cell culture-derived biotechnical products for therapeutic use have risks associated with cell substrate DNA. Regulatory authorities in the USA and Europe have set stringent limits for this contaminant, requiring orders of magnitude reduction from fermenter harvests to purified products. This paper addresses the relevant unit processes (e.g., cell separation, affinity chromatography and ion-exchange chromatography) utilized for purification of mammalian cell culture-derived products as they pertain to removal of DNA contaminants.

Cell Separation↗
PubMed2005

Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR.

Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.

Base Pair Mismatch↗
PubMed1993

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

Bacteria↗
PubMed2005

Use of bleach to eliminate contaminating DNA from the surface of bones and teeth.

The extraction of DNA from archaeological or forensic skeletal remains can provide quite powerful data for analysis, but is plagued by a unique set of methodological problems. One of the most important methodological problems to overcome in such analyses is the presence of modern contamination on the surfaces of bones and teeth, which can lead to false positives and erroneous results unless it is removed before DNA extraction is initiated. Ancient DNA (aDNA) researchers and forensic scientists have employed a number of techniques to minimize such contamination. One such technique is the use of bleach (sodium hypochlorite--NaOCl) to "destroy" contaminating DNA. However, a consensus on the optimum concentration of sodium hypochlorite to be used and the amount of time the bone or tooth should be exposed to it has not emerged. The present study systematically approaches the issue by introducing contamination to ancient bones (from approximately 500 BP) and determining which of several sodium hypochlorite treatments best eliminates surface contamination. The elimination of surface contamination from bone requires immersion in at least 3.0% (w/v) sodium hypochlorite (approximately equal parts of commercial bleach and water) for at least 15 min. Endogenous DNA proved to be quite stable to even extreme sodium hypochlorite treatments (6% for 21 h), suggesting that DNA adsorbs to hydroxyapatite in the bone and that this process facilitates the preservation of DNA in ancient skeletal remains.

DNA↗
PubMed2000

DNA contamination of mortuary instruments and work surfaces: a significant problem in forensic practice?

A study of 20 mortuaries was undertaken to assess whether contamination of instruments, tables and cutting areas due to residual material containing human DNA after routine cleaning is an actual or only a theoretical problem. Of the 20 mortuaries studied, 50% were found to have material containing quantifiable human DNA on the instruments and surfaces sampled. This DNA was amplified and found, in some cases, to have been derived from at least three people. Of those that did not yield measurable amounts of DNA, a number of samples were selected at random, amplified and were found to produce partial profiles indicating the presence of low levels of human DNA. The possible sources of human DNA from mortuaries are discussed as well as means to reduce or irradicate the problem of instrument contamination. Finally the implications of these findings for forensic investigations are discussed.

Autopsy↗