Rapid extraction of DNA suitable for NGS workflows from bacterial cultures using the PDQeX.

Background: PDQeX is a novel, single-step DNA extraction method that purifies nucleic acid from sample in under 30 min. Materials & Methods: Six bacterial suspensions from species with different cell morphologies and growth optima were made. DNA from half the suspension was purified using PDQeX and the other half using a conventional column purification method. Sequencing and analyses using Ion PGM were performed, blinded to extraction method and species. Results: Genomes extracted with either method sequenced successfully. No significant sequence distribution biases were evident between PDQeX and column purification. Surveyed community preference suggested comparable performance between the two extraction methods. Conclusion: DNA prepared using the PDQeX performs as well for whole-genome sequencing as DNA purified using a conventional method, albeit much more rapidly.

An integrated sample-in-answer-out microfluidic chip for rapid human identification by STR analysis.

A fully integrated microfluidic chip for human identification by short tandem repeat (STR) analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-resolution separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (Leff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional instruments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total analysis system for point-of-collection human identification based on genetic analysis.

From sample to PCR product in under 45 minutes: a polymeric integrated microdevice for clinical and forensic DNA analysis.

The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the ß-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the ß-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time.

An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/µL to 7.78 (±1.40)ng/µL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

On the origin and evolution of thermophily: reconstruction of functional precambrian enzymes from ancestors of Bacillus.

Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth, that has been gradually lost over evolutionary time. The genus Bacillus provides an ideal model for studying the evolution of thermophily as it is an ancient taxon and its contemporary species inhabit a range of thermal environments. The thermostability of reconstructed ancestral proteins has been used as a proxy for ancient thermal adaptation. The reconstruction of ancestral "enzymes" has the added advantages of demonstrable activity, which acts as an internal control for accurate inference, and providing insights into the evolution of enzymatic catalysis. Here, we report the reconstruction of the structurally complex core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, E. C. 1.1.1.85) from the last common ancestor (LCA) of Bacillus using both maximum likelihood (ML) and Bayesian inference. ML LeuB from the LCA of Bacillus shares only 76% sequence identity with its closest contemporary homolog, yet it is fully functional, thermophilic, and exhibits high values for k(cat), k(cat)/K(M), and ΔG(‡) for unfolding. The Bayesian version of this enzyme is also thermophilic but exhibits anomalous catalytic kinetics. We have determined the 3D structure of the ML enzyme and found that it is more closely aligned with LeuB from deeply branching bacteria, such as Thermotoga maritima, than contemporary Bacillus species. To investigate the evolution of thermophily, three descendents of LeuB from the LCA of Bacillus were also reconstructed. They reveal a fluctuating trend in thermal evolution, with a temporal adaptation toward mesophily followed by a more recent return to thermophily. Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths. Our results add significant fluctuations to the broad trend in thermal adaptation previously proposed and demonstrate that thermophily is not exclusively a primitive trait, as it can be readily gained as well as lost. Our findings also demonstrate that reconstruction of complex functional Precambrian enzymes is possible and can provide empirical access to the evolution of ancient phenotypes and metabolisms.

Bioremediation of hydrocarbon-contaminated polar soils.

Bioremediation is increasingly viewed as an appropriate remediation technology for hydrocarbon-contaminated polar soils. As for all soils, the successful application of bioremediation depends on appropriate biodegradative microbes and environmental conditions in situ. Laboratory studies have confirmed that hydrocarbon-degrading bacteria typically assigned to the genera Rhodococcus, Sphingomonas or Pseudomonas are present in contaminated polar soils. However, as indicated by the persistence of spilled hydrocarbons, environmental conditions in situ are suboptimal for biodegradation in polar soils. Therefore, it is likely that ex situ bioremediation will be the method of choice for ameliorating and controlling the factors limiting microbial activity, i.e. low and fluctuating soil temperatures, low levels of nutrients, and possible alkalinity and low moisture. Care must be taken when adding nutrients to the coarse-textured, low-moisture soils prevalent in continental Antarctica and the high Arctic because excess levels can inhibit hydrocarbon biodegradation by decreasing soil water potentials. Bioremediation experiments conducted on site in the Arctic indicate that land farming and biopiles may be useful approaches for bioremediation of polar soils.

Hydrocarbon contamination changes the bacterial diversity of soil from around Scott Base, Antarctica.

A combination of culture-independent and culturing methods was used to determine the impacts of hydrocarbon contamination on the diversity of bacterial communities in coastal soil from Ross Island, Antarctica. While numbers of culturable aerobic heterotrophic microbes were 1-2 orders of magnitude higher in the hydrocarbon-contaminated soil than control soil, the populations were less diverse. Members of the divisions Fibrobacter/Acidobacterium, Cytophaga/Flavobacterium/Bacteroides, Deinococcus/Thermus, and Low G+C gram positive occurred almost exclusively in control soils whereas the contaminated soils were dominated by Proteobacteria; specifically, members of the genera Pseudomonas, Sphingomonas and Variovorax, some of which degrade hydrocarbons. Members of the Actinobacteria were found in both soils.

A heteroduplex method for detection of targeted sub-populations of bacterial communities.

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.

A culture-independent survey of the bacterial community in a radon hot spring.

Paralana is an active, radon-containing hot spring situated in a region of South Australia's Flinders Ranges with a long history of hydrothermal activity. Our aim was to determine the bacterial composition of Paralana using a culture-independent, 16S rRNA-based technique. The presence of a diverse bacterial community was strongly suggested by the large number (approximately 180) of different ribotypes obtained upon analysis of nine hot spring samples. DNA sequencing of Paralana 16S rRNA genes corroborated this observation, identifying representatives of seven confirmed and two candidate divisions of the domain Bacteria. These included Cyanobacteria, Proteobacteria (both beta and delta subdivisions), the Cytophaga-Flexibacter-Bacteroides group, Low G + C Gram-positives, Nitrospira, green non-sulfur bacteria, green sulfur bacteria, OP8, and OP12. No known ionizing radiation-resistant Bacteria were identified. Only one Paralana 16S rRNA sequence type (recombinant B5D) was homologous to a sequence previously identified from a radioactive environment.