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PubMed2005

Characterization of a normalized cDNA library from bovine intestinal muscle and epithelial tissues.

Tissue-specific cDNA library sequences (expressed sequence tags, or EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e., microarrays) for gene expression profiling. The objective of this study was to develop and characterize an intestine-specific cDNA library to examine the transcriptome of the bovine gut and identify expressed genes that influence ruminant nutrition and health. We describe BARC-8BOV, a normalized cDNA library developed from mRNA isolated from four distinct intestinal locations (duodenal, jejunal and ileal small intestine, colon) of Holstein dairy cattle resulting in 19,110 5'-EST deposited into the NCBI GenBank EST database. Assembly and clustering of these 19,110 clone sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal species, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not previously represented as EST in nucleotide or protein databases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. Ultimately, these data will also contribute in efforts to annotate the bovine genome.

Animals↗
PubMed2006

The tendency of individuals to transfer DNA to handled items.

This research investigates factors influencing the transfer of DNA to handled objects and the process known as 'shedding'. Volunteers were recruited to hold sterile plastic tubes using experiments originally designed by Lowe et al. [A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Sci. Int. 129 (2002) 25-34]. Transferred cellular material was collected from the tubes and STR profiles generated using the AmpFlSTR SGM Plus multiplex with 28 and 34 PCR cycles. Volunteers were asked to hold the tubes with each hand, and to participate in a series of handwashing experiments. The DNA profiling results obtained from the transferred skin cells were compared. An attempt was made to characterize the volunteers as 'good' or 'bad' shedders and to establish which, if any, of the experimental variables were associated with 'good' shedding. Our results suggest that many factors significantly influence shedding, including which hand an individual touches an item with and the time that has elapsed since they last washed their hands. We have found that it may be more complicated than previously reported to categorise a person as being either a 'good' or a 'bad' shedder and that if truly 'good' shedders exist they may be significantly rarer than some have estimated. In the current research no 'good' shedders were observed in a group of 60 volunteers. Given these results, it seems that rather than being applied to individual forensic cases, knowledge of shedding characteristics will be most useful in providing general background data for the interpretation of trace DNA evidence.

DNA↗
PubMed1989

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2006

Meth-DOP-PCR: an assay for the methylation profiling of trace amounts of DNA extracted from bodily fluids.

Cancer cells release their DNA into the patient's bodily fluids and cancer-specific signatures can be recognized in the circulating DNA. The aberrant methylation of CpG-rich regions in gene promoter sequences is an early marker of cell transformation whose specificity and optimal sensitivity can be achieved by assessing the methylation status of multiple genes ('methylation profiling'). Most of the current technologies for methylation analysis rely upon the combination of chemical conversion of the DNA and PCR analysis for the detection of methylated and unmethylated alleles. However, the small amount of circulating DNA, and its fragmentation, dramatically reduces the template DNA molecules making difficult the methylation profiling. To overcome this limitation, we have developed the Meth-DOP-PCR assay, a combination between a modified degenerate oligonucleotide primed PCR (DOP-PCR) and methylation-specific PCR (MSP), for the high-throughput methylation analysis of trace-amount of circulating DNA. We have demonstrated the concordance between Meth-DOP-PCR and MSP and shown the application of this technique for the methylation analysis of DNA extracted from the serum of lung cancer patients. We have estimated that through this procedure it is possible to obtain at least a 25-fold increase of the number of determinations allowing the methylation profiling from less than 1 ml of serum. Thus, Meth-DOP-PCR appears as a simple, cost-effective and efficient technique, for the development of novel methylation-based diagnostic assays.

Blood Chemical Analysis↗
PubMed2004

Diagnostic of Biomphalaria snails and Schistosoma mansoni: DNA obtained from traces of shell organic materials.

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.

Animals↗
PubMed1999

The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community.

Plasmid genes that are responsible for virulence of Bacillus anthracis are important targets for the DNA-based detection of anthrax. We evaluated the distribution of the Ba813 chromosomal DNA sequence (Ba813) within closely related Bacillus species. Ba813 was systematically identified from 47 strains or isolates of B. anthracis tested, thus indicating its reliability as a tracer for that species. From the 60 strains of closely related Bacillus spp. examined, three bona fide B. cereus and one bona fide B. thuringiensis were found to harbour Ba813. This marker was also detected in Bacillus sp. isolates that were present at high levels in soil samples collected in a place where an anthrax outbreak had occurred. The significance and the possible function of the Ba813 locus is discussed.

Animals↗
PubMed2001

DNA profiling of trace evidence--mitigating evidence in a dog biting case.

A young girl was the victim of a severe dog attack. An animal, suspected of having caused the attack, was later impounded for investigation. Microclots of blood, recovered from the dog's fur, were analyzed by STR DNA. Results showed that this blood was not related to the biting. Other forensic evidence--hairs, fibers, and odontology--failed to connect a particular animal to the attack. The implications of these findings for the dog and its owners are discussed as well as other forensic methods for resolving such cases.

Animals↗
PubMed2006

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces.

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.

Alleles↗
PubMed1996

N,N'-bis[3,3'-(dimethylamino)propylamine]-3,4,9, 10-perylenetetracarboxylic diimide, a dicationic perylene dye for rapid precipitation and quantitation of trace amounts of DNA.

A novel dicationic dye with a polycyclic aromatic perylene core and flexible cationic side chains- N,N'-bis[3, 3'-(dimethylamino)propylamine]-3,4,9,10-perylenetetracarboxylic diimide-termed "DAPER," was synthesized and characterized. The dye appears to exist in a highly stacked form in aqueous solution. DAPER precipitates extremely low concentrations of DNA very rapidly, efficiently, and with a stoichiometry of one tightly bound dye per DNA base pair, corresponding to a neutral complex. Precipitation may occur due to side-by-side association between the polyanionic DNA helix and polycationic dye stacks. DNA precipitation by DAPER is less sensitive to DNA concentration and length, and prevailing salt concentrations, than precipitation with ethanol or propanol. DAPER can be quantitatively extracted from DNA into a standard phenol:chloroform mixture under slightly alkaline conditions. The recovered DNA is suitable for treatment with enzymes typically used in DNA sequencing procedures. The amount of DNA precipitated is accurately determined by visible absorption or fluorescence spectroscopic analyses of the phenol:chloroform extracts. Several samples of DNA can be precipitated, recovered, and quantitated in about 1 h using standard microscale procedures and equipment. The unique qualities of DAPER provide the basis for a very sensitive, rapid, and versatile method for simultaneous precipitation and quantitation of microgram and submicrogram amounts of DNA.

1-Propanol↗
PubMed2000

Voltammetric trace analysis of DNA and RNA.

A voltammetric determination of DNA/RNA is described. The new aspect is the use of the extracellular endonuclease Serratia marcescens in the sample preparation. Using this enzyme it is possible to determine DNA/ RNA with a detection limit of 2-5 pg/mL. This satisfies the requirements of the WHO and the FDA.

Animals↗
PubMed2000

Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA.

The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately.

Africa↗
PubMed2000

Tracing human papillomavirus DNA in nasal polyps by polymerase chain reaction.

Human papillomavirus (HPV) infections are related to the genesis of various benign and malignant human neoplasias. The HPV types 16 and 18 seem to be causally related to the development of most squamous cell carcinoma of the anogenital tract and a proportion of carcinomas of the upper aerodigestive tract. The near 100% positivity of the HPV types 6 and 11 in laryngeal papillomatosis is well established. We investigated whether HPV also plays a role in non-neoplastic mucosal entities such as sinunasal polyposis, the genesis of which has been discussed as being triggered by viral infections. On DNA from 39 sinunasal polyps (33 patients), polymerase chain reaction (PCR) was performed using beta-globin primers for demonstration of amplifiable DNA in the tissue extracts. Consensus primers for the detection of several different HPV types were applied to the beta-globin-positive samples. The results were confirmed by Southern blot hybridization using consensus probes. Cycle sequencing was performed on the positive cases. All 39 samples showed positive signals for beta-globin. HPV-DNA investigations showed a slight positive signal in only 1 of the 39 investigated cases (2.6%). Further molecular investigations of this sample, including cycle sequencing, could not confirm this result. All the other tissue samples remained HPV-DNA-negative. Therefore, those HPV types readily detectable with the PCR primers and probes used are not frequently associated with sinunasal polyposis. The data confirm the hypothesis that HPV is correlated to a lesser extent to infectious mucosal lesions than to proliferative lesions. Furthermore, the results emphasize that the presence of HPV in specific lesions does not occur by chance, but represents a specific infection of the mucosa leading to proliferation and even to malignancy.

DNA, Viral↗
PubMed1993

Studies on DNA adduction with heterocyclic amines by accelerator mass spectrometry: a new technique for tracing isotope-labelled DNA adduction.

DNA adduction in rodents at doses equivalent to human dietary exposure (10(4)-10(6)-fold lower than laboratory studies) is being studied using accelerator mass spectrometry (AMS). AMS is a nuclear physics technique for detection of cosmogenic isotopes and has been used for specifically selecting and counting 14C. Using AMS, DNA adducts are detectable at levels of 1-10 adducts/10(12) nucleotides following acute and chronic dosing regimes with 14C-labelled carcinogens. The adduct detection limit has been imposed by the natural abundance of 14C in the samples and animal-to-animal variation. AMS is also being coupled to HPLC, multidimensional TLC and radio-immunoassay. In addition, AMS's great sensitivity makes it useful for demonstrating that drugs and chemicals do not bind to DNA. The use of AMS, however, is limited to situations where radiolabelled agents can be used. The data suggest that AMS is extremely useful in obtaining quantitative data on the effects of carcinogens on DNA at the low doses common for actual human exposures and may be useful in human studies.

Amines↗
PubMed2002

Delitto Perfetto: foreign DNA disappears without a trace.

A group of NIEHS scientists has recently perfected a new in vivo gene modification technique that enables scientists to quickly generate site-directed mutations onto specific regions of the yeast genome. The researchers were able to create genetic mutations by inserting foreign DNA into host DNA without leaving a trace of the foreign DNA. Dubbed "Delitto Perfetto," Italian for "perfect murder," the new technique leaves behind no clue that foreign DNA was introduced to engineer the desired genetic changes. It could transform the way genetic researchers analyze how genes function and respond to human disease and environmental influences.

Bryopsida↗
PubMed1987

RNA and DNA in melanosomes of hamster melanoma.

The melanosomes were isolated from Syrian hamster melanoma Ma by three different methods. Levels of about 0.2% RNA and less than 0.05% of DNA were detected in the melanosome preparations. The higher the purification of the melanosome samples, the lower the DNA content observed. Consequently, traces of DNA in melanosomes could originate from contamination. The irreversible interaction of DNA with melanosomes in vitro was not demonstrated.

Animals↗
PubMed1996

[Border-crossing source tracing in tuberculosis via DNA fingerprint technique].

OBJECTIVE: Description of the value of modern DNA analysis ('restriction fragment length polymorphism'; RFLP) as a method for mapping tuberculosis transmission. DESIGN: Descriptive. SETTING: Social Health Service Noord-Friesland, the Netherlands. METHOD: A large-scale tuberculosis investigation was conducted in Harlingen in 1993 when a 2,5 year old patient with tuberculous meningitis was reported. Source tracing and contact tracing extended from Harlingen to the west of the Netherlands and even abroad. Modern DNA analysis (RFLP) was used to map the tuberculosis transmission. RESULTS: A total of 6519 persons were screened and 276 infected people were identified, of whom 49 were suffering from active tuberculosis. RFLP analysis showed in 27 of them a 'Harlingen' type Mycobacterium tuberculosis DNA pattern identical to that of the index patient. The source patient was finally traced in England. By the end of 1994 3 more patients were found with the same DNA pattern; their relation with the source patient and the outbreak of tbc remained unrevealed. CONCLUSION: The Harlingen outbreak was extensive and characterized by time-consuming source tracing. Modern DNA analysis proved to be a very useful instrument in identifying the source case.

Adult↗
PubMed2005

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: identification of novel antitumor drug binding site.

The studies of novel inhibitors of DNA topoisomerase I (Topo I) have already become very promising in cancer chemotherapy. Identifying the new drug-binding residues is playing an important role in the design and optimization of Topo I inhibitors. The designed compounds may have novel scaffolds, thus will be helpful to overcome the toxicities of current camptothecin (CPT) drugs and may provide a solution to cross resistance with these drugs. Multiple sequence alignments were performed on eukaryotic DNA topoisomerase I superfamily and thus the evolutionary tree was constructed. The Evolutionary Trace method was applied to identify functionally important residues of human Topo I. It has been demonstrated that class-specific hydrophobic residues Ala351, Met428, Pro431 are located around the 7,9-position of CPT, indicating suitable substitution of hydrophobic group on CPT will increase antitumor activity. The conservative residue Lys436 in the superfamily is of particular interest and new CPT derivatives designed based on this residue may greatly increase water solubility of such drugs. It has also been demonstrated that the residues Asn352 and Arg364 were conservative in the superfamily, whose mutation will render CPT resistance. As our molecular docking studies demonstrated they did not make any direct interaction with CPT, they are important drug-binding site residues for future design of novel non-camptothecin lead compounds. This work provided a strong basis for the design and synthesis of novel highly potent CPT derivatives and virtual screening for novel lead compounds.

Amino Acid Sequence↗