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At least 37 records · Page 2Linked to original sources
PubMed1995

Using chloroplast DNA to trace postglacial migration routes of oaks into Britain.

Postglacial migration is a major factor responsible for the patterns of genetic variation we see in natural populations. Fossil pollen data indicate that early postglacial colonists such as oak, were able to take both western and eastern migration routes into Britain. Analysis at a finer level is now permitted by the use of modern molecular techniques. A 13-bp duplication in the chloroplast tRNA(Leu1) intron occurs in natural populations of East Anglian oaks, but is not found in other parts of Britain or from mainland Europe. The distribution of this marker suggests that the mutation occurred either in southern England, or during migration from the mainland, and became fixed in a source population from which East Anglia was colonized. Planting of non-native trees for roadside boundaries and in the grounds of old houses and estates, explains the absence of the marker from some East Anglian oaks.

Biological Evolution↗
PubMed2003

Random priming PCR strategy to amplify and clone trace amounts of DNA.

Here we report a new methodology to study trace amounts of DNA of unknown sequence using a two-step PCR strategy to amplify and clone target DNA. The first PCR is carried out with a partial random primer comprised of a specific 21-nucleotide 5' sequence, a random heptamer, and a 3' TGGC clamp. The second PCR is carried out with a single 19-nucleotide primer that matches the specific 5' sequence of the partial random primer. Using human and Mycoplasma genitalium DNA as examples, we demonstrated the efficiency of this approach by effectively cloning target DNA fragments from 1 pg DNA sample. The cloning sensitivity could reach 100 fg target DNA templates. Compared to the strategy of first adding adapter sequences to facilitate the PCR amplification of unknown sequences, this approach has the advantage of allowing for the amplification of DNA samples in both natural and denatured forms, which provides greater flexibility in sample preparation. This is an efficient strategy to retrieve sequences from trace DNA samples from various sources.

Cloning, Molecular↗
PubMed2026

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↗
PubMed2003

Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA.

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.

Biotechnology↗
PubMed2000

A multi-state outbreak of tuberculosis among members of a highly mobile social network: implications for tuberculosis elimination.

SETTING: Baltimore, Maryland. OBJECTIVE: To describe a tuberculosis (TB) outbreak among a highly mobile population and the efforts required to control it. DESIGN: Epidemiologic outbreak investigation. RESULTS: Between June 1998 and January 2000, 20 TB outbreak cases were identified, of which 18 were culture-confirmed. Seventeen isolates of Mycobacterium tuberculosis had an identical 11-band DNA fingerprint; another isolate had one additional band and was considered a match. Two cases were diagnosed in New York City; another patient lived primarily in Atlanta, but was diagnosed in Baltimore. Persons in the outbreak were predominantly young (median age 24 years), black, male, infected with the human immunodeficiency virus (HIV), and gay, transvestite or transsexual. Activities common among many TB cases included attending two nightclubs, membership in one of three social 'Houses', attending balls or pageants in East Coast cities, marijuana use, and prostitution. Community outreach, extended contact tracing, DNA fingerprinting, directly-observed therapy, and expanded use of preventive therapy were utilized to assess and control the outbreak. During the outbreak period the Baltimore City TB rate declined by 10%. However, additional public health personnel were required to control the outbreak, resulting in a 17% increase in TB clinic staff. CONCLUSION: As TB rates decline, remaining cases are likely to occur in difficult-to-reach populations. Increased resources per case of TB treated will be required to eliminate TB.

AIDS-Related Opportunistic Infections↗
PubMed2006

Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping.

Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles.

Alleles↗
PubMed2003

Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR.

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k(b) murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 microM primer, a 20-fold increase in the median manufacturer's recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 microg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.

Journal Article↗
PubMed2004

Analysis and interpretation of data from real-time PCR trace detection methods using quantitation of GM soya as a model system.

Recent years have seen an increased interest in DNA trace detection methods involved in many areas of bioanalytical research, such as quantitation of genetically modified (GM) ingredients in food products. There is little in the way of standardisation of data handling from these methods, and the data generated needs to be analysed appropriately if the results are to be interpreted correctly. This paper describes particular aspects of real-time PCR trace detection methods in order to increase the understanding of data generated using this bioanalytical technique. Using the specific example of GM soya detection and quantitation, it focuses on the production of calibration curves based on the mean and individual data values, the interpretation of correlation coefficients, regression techniques, and discusses suitable data analysis arising from simple and more complex experimental designs following transformation. By using the approaches outlined in this paper, more accurate analysis of data from real-time PCR and GM trace detection methods could be achieved.

Calibration↗
PubMed1994

Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification.

AIM: To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples. METHODS: Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. RESULTS: Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA. CONCLUSION: This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available.

Base Sequence↗
PubMed2001

Reprocessing of thermosensitive materials--efficacy against bacterial spores and viruses.

To estimate process parameters for non-thermal methods of antimicrobial inactivation, the half-cycle method is very often used. However, the essential premise of this method of estimation, the independence of microbial inactivation kinetics from the microbial load, seems not to be true. Consequently, the attainment of the sterility assurance level as recommended by the pharmacopoeias by a process which has been validated using the half cycle method is not guaranteed. For the evaluation of such chemo-thermo disinfection processes, the quantification of remaining hepatitis B virus DNA (HBV-DNA) traces on the surface of instruments is a useful tool. An infection can be excluded if a decrease of the HBN3-DNA residues on the instruments below the minimum infective dose can be demonstrated. Using the signal amplification technique to detect HBV-DNA on instruments, the safety of an reprocessing procedures can be improved.

Bacillus subtilis↗
PubMed2001

Trace elements and DNA damage.

A number of studies (mainly in vitro and in vivo animal models) have examined the interaction of trace elements with DNA. Normal dietary levels of various trace elements are required to prevent the occurrence of oxidative damage, and deficiency may increase susceptibility. Conversely, overload of some trace elements, including copper and iron, has been demonstrated to result in adverse effects. However, under normal physiological conditions, such overloads are unlikely to occur.

Antioxidants↗
PubMed2005

Beringian paleoecology inferred from permafrost-preserved fungal DNA.

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.

Cold Climate↗
PubMed2003

The effectiveness of protective clothing in the reduction of potential DNA contamination of the scene of crime.

The use of ultra-sensitive low copy number (LCN) DNA typing allows the analysis of picogram amounts of DNA. Trace evidence accidentally left at a scene of crime (SOC) by the investigating team may be inadvertently collected and analysed, potentially leading to spurious evidence being introduced into the criminal investigation. A series of experiments were undertaken to determine the extent to which an investigator could contribute to any DNA contamination of a scene of crime under different simulated activities. Further, the degree to which any contamination was reduced by the use of commercially available protective clothing was demonstrated. Precautions that should routinely be taken at a scene of crime to reduce the risk of DNA contamination are recommended.

Cough↗
PubMed2001

Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B- to A-form transition on mica.

Achieving the most correct estimate of the contour length of digitized DNA molecules is a key aspect of the microscopic analysis of nucleic acids by either electron microscopy (EM) or atomic force microscopy (AFM). Six different methods, that are mathematically not too complex and suited for common, practical use, have been tested here using simulated polymers in two dimensions and real DNA molecules (564, 1054, 2049 and 4297 bp long) imaged in air by AFM. The main result is that the frequently used Freeman estimator (L(F) = n(e) + square root 2n(o)) overestimates the real contour length of the polymers by about 4%. More accurate estimates are obtained with the Kulpa estimator (L(K) = 0.948n(e) + 1.343n(o)) or with the corner count estimator (L(C) = 0.980)n(e) + 1.406n(o) - 0.091n(c)). In the range of the DNA sizes and magnifications we have considered, however, the best results are obtained with an ad hoc developed routine that smoothes the DNA trace by a polynomial fitting of degree 3 over a moving window of 5 points. Under these conditions, the difference between the measured and the real contour length of the molecules is less than 0.4%. The accuracy of this procedure allowed us to reveal a discrete, size-dependent, shortening of DNA molecules deposited onto mica under low salt conditions and imaged in air by AFM. Awareness of this structural alteration, that can be attributed to a partial transition from B- to A-form DNA, may lead to a more correct interpretation of DNA molecules or protein-DNA complexes imaged by AFM.

Algorithms↗
PubMed1996

Use of IS6110 DNA fingerprinting in tracing man-to-man transmission of Mycobacterium tuberculosis in the Czech Republic.

The epidemiological relationship between tuberculosis cases in a prison and between cases within five families was investigated. Therefore, the isolated Mycobacterium tuberculosis strains were subjected to restriction fragment length polymorphism (RFLP) analysis using insertion sequence IS6110 as a probe. In case of 11 patients, the expected links of transmission were confirmed by RFLP typing. In contrast, in case of 4 patients the conclusion of classical contact tracing were not in agreement with the DNA fingerprinting results. These findings reinforce the usefulness of this recently developed technique as an additional tool in contact tracing. The IS6110DNA fingerprints of all strains investigated consisted of 7 to 13 bands and showed a high degree of polymorphism. Comparison of these fingerprints with those recorded in the Czech Republic previously, revealed the presence of a predominant DNA fingerprint type, without a known connection between the cases. Furthermore, other patterns found in the present study showed a high degree of similarity with the previously obtained fingerprints. Part of the patients were sampled twice. All of these double isolates showed identical fingerprints, confirming the previously described stability of IS6110DNA fingerprints. In contrast, from one couple of strains, isolated from husband and wife both suffering from tuberculosis, a slight change in one of the two patterns was observed. The patterns shared 10 band positions, confirming the expected relationship between these cases, but one of the patterns contained one additional band.

Contact Tracing↗