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A rare and atypical case of long-distance indirect DNA transfer: Contamination from an investigator never present at the scene.

To maximize the usefulness of DNA obtained from biological samples in forensic genetics, it is crucial to avoid DNA contamination throughout all procedures, from sample collection at crime scenes to STR profile generation in DNA laboratories. This study reports a rare and atypical case of DNA contamination in a forensic setting. During the analysis of biological evidence from a cold case preserved for 18 years, the STR profile obtained from the surface of a plastic bag matched that of an investigator, identified through the DNA elimination database. Case reconstruction confirmed that the investigator-who was located 80 km from the DNA laboratory and had never entered the crime scene or the sample storage room-was not a suspect and that the obtained STR profile originated from contamination. The most plausible explanation for the contamination was indirect transfer: investigator's DNA had adhered to a colleague's clothing and was subsequently dislodged and deposited onto the surface of the plastic bag as the colleague approached the sample pretreatment area. This study integrates trace DNA profiling of challenged samples with rapid contamination investigation and proposes prevention and control measures. This case underscores that, although DNA is widely regarded as the "gold standard" in forensic genetics, its interpretation must be considered within the context of the entire case. Conclusions should not be drawn based solely on a single DNA result.

Humans

Dextran sulfates as a contaminant of DNA extracted from concentrated viruses and as an inhibitor of DNA polymerases.

Dextran sulfate is commonly used with polyethylene glycol to concentrate viruses before extraction of their DNA. However, dextran slulfate then easily contaminated such DNA and acted as a potent inhibitor of DNA polymerases from Bacillus subtilis (III), phage PBS2, and phage T4. Dextran sulfate only weakly inhibited Micrococcus luteus and Escherichia coli DNA polymerase I preparations.

Bacteriophages

[Absence of anti double stranded DNA antibodies in viral hepatitis (author's transl)].

The presence of anti double stranded DNA antibodies (ds DNA Ab) in the sera of patients with acute viral hepatitis and B virus chronic active hepatitis has recently been described. In contrast with these results we found no ds DNA ab, but anti single stranded DNA antibodies (ss DNA ab) in the sera from patients with these diseases. The ds DNA ab were studied simultaneously by two methods: Immunofluorescence (IF) on crithidia luciliae and Farr test. In this later case it was shown that the ds DNA preparation contained no ss DNA because it did not react with an anti ss DNA rabbit serum. Our results indicate that the above mentioned authors have been using in their Farr test a preparation of ds DNA contaminated by ss DNA, thus revealing the presence of anti ss DNA ab but not anti ds DNA ab, in the sera of their patients. On the other hand the presence of ds DNA ab has been shown in the sera of 85% of the patients with systemic lupus erythematosus and of 20,5% of the patients with non EB chronic active hepatitis. At least one part of these later are probably autoimmune diseases.

Acute Disease

Purification of plasma membrane of guinea pig peritoneal macrophages.

A method using sucrose density gradient centrifugation is described for the purification of plasma membrane of guinea pig peritoneal exudate macrophages. Assays for composition and for marker enzyme activities have been modified for use with the small amounts of subcellular macrophage material. The plasma membrane was obtained in 57% yield and contained 7% of the protein. The purified plasma membrane fraction is five-fold enriched in phospholipid to protein ratio and contains no contaminating DNA, none of the cytoplasmic marker lactate dehydrogenase, no detectable mitochondrial contamination and a low contamination with lysosomal enzymes (7%). Purified plasma membrane containing 4 mg of protein can be prepared from 1 ml of pelleted macrophages in a one-day operation.

Animals

DNA sequencing for microbial surveillance in cystic fibrosis airways: advances, challenges, and clinical translation.

SUMMARYDNA sequencing has revolutionized microbial surveillance in cystic fibrosis (CF), transforming pathogen identification from culture-dependent to total microbial community identification using molecular-based approaches. Techniques such as 16S rRNA gene sequencing have uncovered the complexity of the CF airway microbiome, while shotgun metagenomics, metatranscriptomics, and viromics now provide strain-level, functional, and viral insights beyond bacterial identification. Despite these advances, key technical and logistical challenges remain, including the processing of high-viscosity sputum samples, overwhelming host DNA contamination, managing large data sets, and the integration of complex bioinformatic outputs into clinical workflows. Emerging innovations such as host DNA depletion protocols, targeted enrichment panels, and adaptive sampling on Oxford Nanopore platforms are helping to overcome these barriers, improving microbial recovery and sequencing efficiency. As cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies are changing the lives of people with cystic fibrosis (pwCF), sequencing offers an unprecedented opportunity to track potential microbial adaptation in response. This review investigates current advances, limitations, and translational opportunities in DNA sequencing for CF airway microbiome surveillance, highlighting how these technologies can help reshape research and clinical microbiology in the post-modulator era.

Cystic Fibrosis

FSH stimulation of DNA synthesis in Sertoli cells in culture.

The incorporation of [2H]thymidine into nuclear DNA was investigated in cultured Sertoli cells prepared from testes of 20-day-old rats. Addition of follicle-stimulating hormone (FSH) or dibutyryl cyclic, 3',5'-adenosine monophosphate (DBCAMP) to the culture medium greatly increased incorporation, expressed either as total amounts of [3H]thymidine incorporated per mug DNA or as the percentage of Sertoli cells with labeled nuclear DNA. No stimulation was observed in cells cultured in the presence of testosterone, insulin or cyclic 3',5'-GMP (cGMP). Light and electron microscopic autoradiographic analysis was employed to establish the identity of Sertoli cells having labeled nuclear DNA. Contaminating spermatogonia, which also took up labeled [3H]thymidine, were excluded from cell counts. In addition, Sertoli cells prepared from testes of irradiated 20-day-old germinal cell depleted rats were also observed to incorporate more [3H]thymidine into nuclear DNA when cultured in a chemically defined medium in the presence of FSH. DNA synthesis was abolished by prior treatment of cells with cytosine arabinoside. In separate experiments, the incorporation of [3H]thymidine into DNA of peritubular myoid cells was shown to be independent of FSH or dbcAMP.

Animals

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. II. Characterization of pre-mRNA isolated by phenol extraction and poly(U)-Sepharose chromatography.

Precursor mRNA (pre-mRNA) was extracted from erythroid enriched bone marrow cells of the rabbit by the methods of Georgiev and Mantieva modified by Markov and Arion and of Holmes and Bonner, respectively. Density gradient centrifugation, base analysis and the effects of alpha-amanitin and actinomycin D on the synthesis of the cellular RNA showed signs of degradation in the rRNA-free 85 degrees C-fraction of the preparation according to Georgiev and Mantieva and a substantial rRNA contamination of the 65 degrees C-fraction. This RNA-fraction as well as the total RNA-preparation extracted according to Holmes and Bonner was purified from rRNA by affinity chromatography on poly(U)-Sepharose. Poly(A)+-RNA of all size-classes, among it a substantial amount of high molecular weight RNA (greater than 45 S), was isolated by this purification procedure. Especially the extraction according to Holmes and Bonner yields high molecular weight material but the critical step of this procedure often resulting in degradation of the RNA is the DNase treatment of the heavily DNA-contaminated total RNA-preparation either due to RNase contamination of the DNase or to the existence of RNase in the less intensive deproteinized RNA. The investigated cellular system is characterized by a very intensive rRNA synthesis which is typical for cells in the early stages of hematopoiesis. In contrast to investigations with purified RNA-polymerases and subcellular systems, but in accordance with data of in vivo experiments, alpha-amanitin inhibits both the pre-mRNA and the pre-rRNA synthesis.

Animals

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.

Adenoviridae

Isolation and characterization of germ line DNA from mouse sperm.

Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas.

Animals

Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells.

The in vivo effects of ethidium bromide on the integrity of mitochondrial DNA have been studied in a mouse L-cell system in which this DNA may be nearly exclusively radiolabelled. This allows the detection of mitochondrial DNA in the presence of contaminating nuclear DNA and eliminates the need for extensive purification of mitochondria or the use of deoxyribonuclease. The mitochondrial DNA in treated cells rapidly attains a high negative superhelix density and is not substantially nickel or degraded over the course of several days.

DNA, Mitochondrial

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.

Coliphages

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength.

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.

Animals

Short-read genome skimming enables molecular barcoding of old myxomycete collections.

This study evaluates the effectiveness of Illumina-based genome skimming for barcoding myxomycete herbarium collections ranging from 29 to 91 years in age. We successfully retrieved partial sequences of the standard marker gene (nucSSU) in all cases, as well as additional markers (mtSSU, EF1a, and COI) for certain collections. Altogether, 28 genes were recognized in the studied material. In a 33-year-old specimen of Lindbladia tubulina, the assembly reached an N50 of 4.19 kb, enabling the recovery of extended functional loci. The input genomic DNA quantity emerges as the primary determinant of sequencing success. Samples with high DNA yields provide representative amounts of contigs coming confirmedly (matching sequences in the NCBI nucleotide database) or potentially (no-hit fraction) from myxomycetes, regardless of specimen age. In addition to target DNA, we revealed distinct signals of both anthropogenic contamination (human DNA and skin microflora) and natural substrate inhabitants, including oribatid mites and bacteria from dead wood, soil, and grass litter. Thus, even in old collections, metagenomic data still carry information regarding the substrate upon which the myxomycete developed. The results demonstrate that short-read genome skimming may help to integrate historical type material of myxomycetes into contemporary phylogenetic research. This method overcomes the length-dependent limitations of traditional Sanger sequencing, thus providing a roadmap for the future of museomics in myxomycetology.

Amoebozoa

Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells.

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.

Animals

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.

Animals

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

Filtration and immunoprecipitation in the elimination of DNA polymerase activity associated with bacterial contamination of sera positive for hepatitis B e antigen and its corresponding antibody.

Samples of serum inoculated with Escherichia coli and serum that became contaminated with bacteria after exposure to a laboratory atmosphere demonstrated elevated DNA polymerase activity. The levels of activity were well within the range of values found in hepatitis B e antigen (HBeAg)-positive samples. The bacterial polymerase activity was markedly reduced by a single passage of serum samples through a 0.22-micron Millipore filter prior to analysis. Repeated filtration did not result in a substantial further decrease in polymerase activity. In sera that were heavily contamined with E. coli, however, filtration was not successful in reducing bacteria-associated polymerase activity to a base-line uncontaminated level. In such instances double antibody immunoprecipitation proved effective in elimination of bacterial activity. When bacterial contamination of serum samples is a possibility, specimens should be subjected to either Millipore filtration or immunoprecipitation prior to analysis, particularly when correlation of DNA polymerase activity with HBeAg and its corresponding antibody is attempted.

Antibodies, Viral