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Le Roux D.,University of Versailles | Root B.E.,MicroLab Diagnostics Inc | Hickey J.A.,MicroLab Diagnostics Inc | Scott O.N.,MicroLab Diagnostics Inc | And 6 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2014

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. © 2014 the Partner Organisations.

Lounsbury J.A.,University of Virginia | Coult N.,University of Virginia | Miranian D.C.,University of Virginia | Cronk S.M.,University of Virginia | And 5 more authors.
Forensic Science International: Genetics | Year: 2012

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. © 2012 Elsevier Ireland Ltd. All rights reserved.

Lounsbury J.A.,University of Virginia | Coult N.,University of Virginia | Kinnon P.,ZyGEM Corporation | Saul D.,ZyGEM Corporation | Landers J.P.,University of Virginia
14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 | Year: 2010

Historically, most microfluidic devices for genetic analysis have been fabricated in glass, a substrate that is costly and, if not regenerated properly between analyses, can have problems with cross-contamination. The use of polymeric substrates, such as poly(methylmethacrylate) (PMMA), allows for simplified fabrication with the possibility for inexpensive and disposable microdevices. The work presented in this paper demonstrates the use of plastic microdevices for DNA extraction and amplification of buccal swabs applicable to forensic short tandem repeat (STR) analysis, and represents advancement towards less expensive, integrated, disposable devices.

Chomic A.,Lincoln University at Christchurch | Pearson M.N.,University of Auckland | Clover G.R.G.,Health Diagnostic Laboratory | Farreyrol K.,University of Auckland | And 3 more authors.
Plant Pathology | Year: 2010

This study, using RT-PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low-degeneracy generic primers with RT-PCR to amplify 68-, 75- and 129/156-bp regions of the Luteoviridae coat-protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV). -PAV, BYDV-PAS, BYDV-MAV (129 and/or 156 bp amplicons). , Soybean dwarf virus, Bean leafroll virus (68 bp amplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid-borne yellows virus, Cereal yellow dwarf virus-RPV (68-bp amplicon) and Sugarcane yellow leaf virus (75-bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus-1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection-detection tool of benefit to biosecurity authorities in nursery-stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as-yet undiscovered species within the Luteovirus and Polerovirus genera. © 2010 The Authors.

Hobbs J.K.,University of Waikato | Shepherd C.,University of Otago | Saul D.J.,ZyGEM Corporation Ltd. | Demetras N.J.,University of Waikato | And 4 more authors.
Molecular Biology and Evolution | Year: 2012

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. 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. © The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

ZyGEM Corporation Ltd | Date: 2011-04-27

The present invention relates to the preparation of a method for the preparation of nucleic acid samples, including the steps of adding at least one non-specific thermophilic enzyme to a sample containing nucleic acid for testing, and incubating said sample for a preferred period at 65-80 C. as required to effect one or more of lysis of cells, digestion of proteins, digestion of cell-wall enzymes, via activity of said thermophilic enzyme, wherein said thermophilic enzyme is substantially stable and active at 65-80 C. but which is readily autolysed and/or denatured when said sample is incubated at or above 90 C.

Zygem Corporation Ltd. and Lockheed Martin | Date: 2014-03-27

A cartridge interface module (CIM), configured to engage with a removable microfluidic cartridge in a nucleic acid analyzer system can include a fluidics component, which is configured to initiate and support a liquid extraction of nucleic acids from a biological sample contained in the removable microfluidic cartridge. The CIM also includes a polymerase chain reaction (PCR) assembly component which can be configured to initiate and support amplification of the extracted nucleic acids. The CIM may also include a high voltage electrodes component that is configured to initiate and support separation of the amplified nucleic acids into nucleic acid fragments in a separation channel of the removable microfluidic cartridge. The CIM also includes a detection optics component that can be configured to collect, detect, and direct label nucleic acid fragments. The CIM is configured to integrate with a microfluidic chip architecture of an inserted removable microfluidic cartridge.

Zygem Corporation Ltd. | Date: 2012-07-16

Aspects of the disclosure provide a microfluidic chip to facilitate DNA analysis. The microfluidic chip includes a first domain configured for polymerase chain reaction (PCR) amplification of DNA fragments, a dilution domain coupled to the first domain to dilute a PCR mixture received from the first domain, and a second domain that is coupled to the dilution domain so as to receive the amplified DNA fragments. The second domain includes a separation channel that is configured to perform electrophoretic separation of the amplified DNA fragments. In addition, the disclosure provides a DNA analyzer to act on the microfluidic chip to perform an integrated single chip DNA analysis.

ZyGEM CORPORATION LTD. and Lockheed Martin | Date: 2013-12-04

A microfluidic chip includes one or more reaction chambers to hold fluids for chemical or biochemical reactions, such as PCR. A non-contact heat source heats the reaction chamber and the fluid, such that the heat source does not contact the reaction chamber or the fluid. The heat source can heat the reaction chamber and the fluid separately, where the reaction chamber and the fluid separately absorb heat radiation from the heat source. A temperature sensor acquires a temperature of the reaction chamber and/or the fluid. Control circuitry controls the heat source according to a cycling profile for the reaction in the fluid to cycle the heat source between heating and not heating the reaction chamber and the fluid based on the temperature acquired by the temperature sensor. Cooling can be provided passively or actively.

Zygem Corporation Ltd | Date: 2016-08-09

Chemical preparations containing enzymes for scientific purposes, namely, for agricultural purposes, or DNA analysis, or bio-security purposes or forensic purposes; chemical preparations for scientific purposes, namely, for agricultural purposes, or DNA analysis, or bio-security purposes, or forensic purposes; diagnostic preparations other than for medical or veterinary purposes; chemical reagents for non-medical purposes; enzymes and buffers for non-medical scientific and research purposes, namely, for agricultural purposes, or DNA analysis, or bio security purposes or for forensic purposes; chemical preparations for scientific purposes, namely, dilution solutions for diluting nucleic acids, buffering nucleic acids, or preserving nucleic acids and used for non-medical purposes, namely, for agricultural purposes, or DNA analysis, or bio-security purposes, or forensic purposes; chemical preparations for scientific purposes, namely, chemical preparations which facilitate the release of nucleic acids, other than for medical or veterinary purposes.

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