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At least 667 records · Page 37Linked to original sources
PubMed1995

Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials.

A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals. Defined pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. The overall process yield of purified plasmid DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli DNA levels are reproducibly below 1% as measured by Southern analysis. Endotoxin levels are less than 0.03 endotoxin units/micrograms plasmid DNA and residual protein is undetectable. This process was used to produce 100 mg of VCL-1005 for use in an active clinical protocol.

Blotting, Southern↗
PubMed1996

A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus.

Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.

Cloning, Molecular↗
PubMed2003

A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking.

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.

Animals↗
PubMed2005

A simple automated instrument for DNA extraction in forensic casework.

The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.

Blood Stains↗
PubMed1983

Absence of maternal contamination of chorionic villi used for fetal-gene analysis.

Chorionic villi can be obtained by direct transcervical aspiration at 9 to 10 weeks' gestation and used for analysis of fetal DNA. However, for the method to be reliable, there must be no detectable contamination by maternal DNA. To investigate the question of contamination, we compared the DNA of chorionic villi from five fetuses with that obtained from maternal lymphocytes, using the restriction endonuclease Taql and specific DNA probes for a pair of alleles on the X chromosome. The alleles yield fragments of different lengths when digested with Taql (length polymorphism), which can be demonstrated by electrophoresis and hybridization with the radioactive DNA probes. If the pattern obtained with the chorionic DNA is different from that obtained with the maternal DNA, contamination is not present. In two cases the fetal DNA of the chorionic villi was shown to be uncontaminated by maternal tissue. In one of these cases the mother was heterozygous and the fetus was homozygous; in the other the mother was homozygous and the fetus was heterozygous. In three other cases no definitive conclusions could be drawn, because the genotypes of the fetus and mother were identical. We conclude that chorionic villi at 9 to 10 weeks' gestation are a source of fetal DNA that can be used for gene analysis, with no detectable contamination by maternal DNA.

Alleles↗
PubMed1988

Application of Y chromosomal repetitive sequences to sexing mouse embryos.

Identification of sex is often necessary to evaluate genetic or teratogenetic effects on embryonic development. A simple molecular technique to identify the sex of mouse embryos was studied using a Y chromosomal repetitive sequence (designated 145SC5). Since this technique does not require purification of DNA, it is particularly suitable for processing many embryos. Furthermore, 145SC5 detects 1% contamination of male DNA in a male-female DNA mixture. These results suggest that 145SC5 is a powerful molecular tool in a variety of studies on mouse development.

Animals↗
PubMed1993

Tri-primer PCR can distinguish free and cloned viral DNA in a single reaction.

A polymerase chain reaction based method is described which distinguishes free and cloned JC virus (JCV) DNA in a single reaction. The method utilizes three primers and amplifies DNA fragments of two different sizes, one specific for free and another specific for pBR322 cloned viral DNA. The procedure was used to simultaneously detect the presence of free and contaminating cloned JCV DNA in brain tissues.

Base Sequence↗
PubMed2004

MtDNA sequencing from zooplankton after long-term preservation in buffered formalin.

Molecular genetic analysis of zooplankton has been slowed by the usual practice of preservation and storage of samples in dilute formalin solutions, which are not always adequately buffered for pH. We report here the determination of DNA sequences for Meganyctiphanes norvegica (Crustacea, Euphausiacea) preserved and stored in buffered formalin for up to 25 years. Specifically designed molecular protocols for DNA extraction and PCR amplification yielded valid sequence data for short (approximately 100-200 bp) regions of the mitochondrial cytochrome b (mtCYB) gene for individual euphausiids. Critical aspects of our approach include: extended extraction and proteinase-K digestion to maximize DNA yield; use of protocols requiring short DNA fragments; design of species-specific PCR primers to minimize risks of contamination by exogenous DNA; and comparison with published DNA sequences for the same gene and species. We conclude that the yield of DNA and the success of subsequent molecular analyses depend primarily on the length of time the tissue has been exposed to formalin and the pH of the solution. Zooplankton samples intended for molecular analysis should preferably be preserved and maintained in ethanol or deep-frozen, but long-term storage in buffered formalin does not preclude some types of molecular genetic analysis.

Animals↗
PubMed1998

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR.

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10(5)-2 x 10(5)) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1-4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.

Cell-Free System↗
PubMed2001

Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma.

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.

Alleles↗
PubMed1987

Investigation of association of mycobacteria with inflammatory bowel disease by nucleic acid hybridization.

We used DNA-DNA hybridization to characterize a mycobacterial isolate, strain Linda, that was obtained from a patient with Crohn's disease and that has been reported to cause ileitis in experimental animals. We also investigated the association of this mycobacterium with Crohn's disease. Our results identified Mycobacterium strain Linda as a strain of Mycobacterium paratuberculosis, the etiologic agent of Johne's disease, a disease of ruminants that has some superficial resemblance to Crohn's disease. Sequences that hybridized with strain Linda DNA probes were detected in DNA extracted from human intestinal tissues from patients with Crohn's disease, ulcerative colitis, and noninflammatory bowel disease. These hybridizing DNA sequences were more prevalent in the muscle layers than in the intestinal mucosa, making it unlikely that they represented DNA from bacterial contaminants in the intestinal lumen. Measurement of the melting temperatures of the DNA-DNA hybrids formed between strain Linda probes and tissue DNAs indicated that the related sequences detected were of mycobacterial origin but were not identical to each other or to strain Linda DNA. These results do not support the proposed specific relationship between Mycobacterium strain Linda and Crohn's disease. The possible etiologic role of mycobacteria in Crohn's disease is discussed.

Colitis, Ulcerative↗
PubMed1992

Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses.

Reverse transcription of the retroviral genome is thought to start after virions enter target cells. Purified preparations of human immunodeficiency virus were found to contain virus-specific DNA, detectable by polymerase chain reaction amplification. This DNA resulted from reverse transcription in newly assembled virus particles and not from contamination by cellular DNA, because virions contained a striking excess of early versus late transcripts and because the accumulation of these products was sensitive to 3'-azido-3'-deoxythymidine (zidovudine) treatment of producer cells. A similar observation was made with murine amphotropic retrovirus particles. It is therefore likely that all retroviruses contain partial reverse transcripts.

Animals↗
PubMed1995

Validation and population studies of the loci LDLR, GYPA, HBGG, D7S8, and Gc (PM loci), and HLA-DQ alpha using a multiplex amplification and typing procedure.

Studies were performed to evaluate the forensic applicability of multiplex amplification of the loci low density lipoprotein receptor, glycophorin A, hemoglobin G gammaglobin, D7S8, and group-specific component (PM loci) and simultaneous typing of these loci using a reverse dot blot approach where allele specific oligonucleotide probes are immobilized on a nylon membrane strip. These results were obtained by using the AmpliType PM PCR Amplification and Typing Kit. The experiments included: mixed body fluid studies; chemical contaminant effects on the DNA in body fluid samples; the effect of typing DNA from body fluid samples deposited on various substrates; the effect of microorganism contamination on typing DNA derived from blood and semen; the effect of sunlight and storage conditions on DNA typing; determination of the sensitivity of detection of the PM test kit; determination of cross-reactivity of DNA from species other than human; typing DNA derived from various tissues from an individual; and an evaluation of the hybridization temperature of the assay. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable PM typing results. Allele and genotype frequencies for six loci (PM loci and HLA-DQ alpha) were determined in African Americans. Caucasians, southeastern Hispanics, and southwestern Hispanics. All loci meet Hardy-Weinberg expectations and there is little evidence for association of alleles between the loci. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple locus DNA profile in various general United States populations.

Alleles↗
PubMed1997

[Development and application of the method for controlling contamination of tumor samples with normal cells to detect deletion of P16 gene in primary lung carcinoma].

OBJECTIVE: The method is to be established to effectively promote the sensitivity of the detection of P16 gene deletion from tumor tissues mixed with normal cells and to determine the relationship between the gene alteration and the lung cancers. METHODS: The normal DNA and that of MCF-7 cells with P16 gene deletion was proportionally mixed and the mixture was limitedly diluted. The exon2 of P16 gene and internal control gene fragment were amplified with template of various amounts of DNA. And polymerase chain reaction (PCR) technique was used in the amplification of exon2 of P16 gene with certain amount of lung cancer DNA as the templates. The amplified fragment was analyzed with SSCP. RESULTS: Normal DNA did not contaminate P16 gene deletion when DNA template was 5 ng and normal cells less than 40%. No deletion and mutation of P16 gene was found in the small cell lung cancer and normal cancer-surrounding lung tissues, while P16 gene deletion and mutation were found respectively in 21 and 3 cases of 52 cases of non-small cell lung cancer. The rate of P16 gene deletion (72%) in lung cancer with lymphatic involvement was significantly higher than that in lung cancer without the metastasis (16.8%) (P < 0.05). It is found that P16 gene deletion was markedly associated with pathologic manifestation and prognosis of non-small cell lung cancer (P < 0.05). CONCLUSIONS: Controlling of the amount of template DNA in PCR helps to promote the sensitivity of detection of P16 gene deletion. The abnormality of P16 gene might relate to the malignant development of non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed2005

Isolation and analysis of DNA from archaeological, clinical, and natural history specimens.

The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. Novel methods exist for the isolation, purification, and analysis of aDNA because these DNA templates are likely to be damaged, fragmented and?or associated with non-nucleic acid material. However, contamination of ancient specimens and DNA extracts with modern DNA is more widespread than is generally acknowledged and remains a significant problem in aDNA analysis. Studies of human aDNA are uniquely sensitive to contamination due to the continual presence of potential contamination sources. Meticulous authentication of results and careful selection of polymorphic markers capable of distinguishing between aDNA and probable DNA contaminants are critical to a successful aDNA study.

Archaeology↗
PubMed1991

A rapid method of sample preparation for detection of DNA viruses in human serum by polymerase chain reaction.

A rapid method of serum treatment is described that can be used for the detection of viral DNA by polymerase chain reaction (PCR). The key feature of the assay is inactivation of inhibitory serum factors by controlled heating of serum. This method avoids DNA extraction. It is very fast and limited sample handling decreases the chances of contamination by exogenous DNA. It has been successfully used for demonstration of parvovirus B19, hepatitis B virus, and human cytomegalovirus DNA in patient sera. Since pathological components in sera from patients with various diseases do not interfere in the assay, it can be used as a sensitive and safe screening assay for DNA viruses in a routine clinical setting.

Base Sequence↗