The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

Sampling tissue for DNA analysis of trees: Trunk cambium as an alternative to canopy leaves

The number of studies of tropical tree species that use molecular tools is increasing, most of which collect leaf tissue for genomic DNA extraction. In tropical trees the canopy is not only frequently inaccessible, but also, once reached, the leaf tissue is often heavily defended against herbivory by high concentrations of anti-predation compounds, which may inhibit downstream applications, particularly PCR. Cambium tissue, accessed directly from the tree trunk at ground level, offers a readily accessible resource that is less hampered by the presence of defensive chemicals than leaf tissue. Here we describe a simple method for obtaining tissue from the cambial zone for DNA extraction and test the applicability of the method in a range of tropical tree species. The method was used successfully to extract DNA from 11 species in nine families. A subset of the DNA extracts was tested in more detail and proved to be highly suitable for AFLP analysis.

Evidence for sensitisation of DNA to oxidative damage during isolation

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.

A importância do DNA na Medicina Dentária Forense

Com o presente trabalho pretende-se discutir a importância do DNA na resolução de casos de investigação forense. O Homem, desde os tempos mais remotos tem revelado interesse na confirmação da identidade dos seus semelhantes, pelo desenvolvimento e prática de diversas técnicas de identificação. A partir de uma revisão da literatura, fez-se o estudo dos métodos de identificação humana, sendo este mais aprofundado no que diz respeito ao DNA. Frequentemente são encontrados corpos em que a única peça disponível para o processo de identificação é o dente. As peças dentárias são as estruturas mais resistentes e estáveis do corpo humano, mantendo as suas características e propriedades por longos períodos de tempo pós-morte. A análise do DNA contribui de forma muito importante para os processos de reconhecimento humano, principalmente em casos em que outros métodos de identificação falham devido a decomposição, fragmentação, incineração ou inexistência de dados comparativos ante-morte. Para a obtenção de um perfil genético é necessário executar as seguintes etapas: extração do DNA, sua quantificação e amplificação seguida de análise de determinadas regiões do genoma. Nos dias de hoje, muitos casos de identificação necessitam de uma abordagem multidisciplinar, isto porque em algumas situações existe falta de material padrão ou, por outro lado, pode haver mais do que uma evidência a ser examinada.

Extracting DNA from ‘jaws’: high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the β-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the β-actin and 28S markers, with the exception of Sus scrofa (pig) β-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Gene amplified in oesophageal cancer 1 (GAEC1) amplification in colorectal cancers and its impact on patient's survival

GAEC1 (gene amplified in oesophageal cancer 1) is located at 7q22.1, first identified in oesophageal cancer.1 Initial work indicated that GAEC1 can act as an oncogene.2 Our pilot study found ∼80% of colorectal cancers showing amplification of GAEC1.3 In this research, we will study GAEC1 copy number in colon cancer cell lines and colorectal tissues, and its prognostic significance. Two human colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were obtained from American Type Culture Collection. Culturing conditions for these cell lines were as published previously.4 Tissues were collected from 283 patients (213 Australian; 70 Japanese) diagnosed with colorectal cancers. Ninety surgically removed non-cancer colorectal tissues (diverticular diseases, hyperplastic polyps and volvulus) were used as controls. H&E stained sections from each cancer were checked to select a block with sufficient cancer tissue and representative morphological features for each patient for DNA extraction...

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Single-walled carbon nanotube-based polymer monoliths for the enantioselective nano-liquid chromatographic separation of racemic pharmaceuticals

Many protocols have been used for extraction of DNA from Thraustochytrids. These generally involve the use of CTAB, phenol/chloroform and ethanol. They also feature mechanical grinding, sonication, N2 freezing or bead beating. However, the resulting chemical and physical damage to extracted DNA reduces its quality. The methods are also unsuitable for large numbers of samples. Commercially-available DNA extraction kits give better quality and yields but are expensive. Therefore, an optimized DNA extraction protocol was developed which is suitable for Thraustochytrids to both minimise expensive and time-consuming steps prior to DNA extraction and also to improve the yield. The most effective method is a combination of single bead in TissueLyser (Qiagen) and Proteinase K. Results were conclusive: both the quality and the yield of extracted DNA were higher than with any other method giving an average yield of 8.5 µg/100 mg biomass.

Utility of temporal artery biopsy samples for genome-wide analysis of giant cell arteritis

Giant Cell Arteritis (GCA) is the most common vasculitis affecting the elderly. Archived formalin-fixed paraffin-embedded (FFPE) temporal artery biopsy (TAB) specimens potentially represent a valuable resource for large-scale genetic analysis of this disease. FFPE TAB samples were obtained from 12 patients with GCA. Extracted TAB DNA was assessed by real time PCR before restoration using the Illumina HD FFPE Restore Kit. Paired FFPE-blood samples were genotyped on the Illumina OmniExpress FFPE microarray. The FFPE samples that passed stringent quality control measures had a mean genotyping success of >97%. When compared with their matching peripheral blood DNA, the mean discordant heterozygote and homozygote single nucleotide polymorphisms calls were 0.0028 and 0.0003, respectively, which is within the accepted tolerance of reproducibility. This work demonstrates that it is possible to successfully obtain high-quality microarray-based genotypes FFPE TAB samples and that this data is similar to that obtained from peripheral blood.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction.

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.