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Report on Qiagen Columns with Precipitation versus Packed Bed Technology for Trace Amounts of DNA

Description: The assured limit of detection (LOD), where 100% of the PCR assays are successful, for the Qiagen spin column is dramatically improved when combined with an ethanol precipitation step of the eluted sample. A detailed SOP for the ethanol precipitation was delivered as a separate report. A key finding in the precipitation work was to incubate the ethanol precipitation at -20{sup o}C overnight when concentrating low copy number samples. Combining this modified ethanol precipitation with the Qiagen spin columns, the limit of assured detection was improved by 1-2 orders of magnitude, for the aliquot and assay variables used. The lower limit of detection (defined as when at least 1 assay of 1 aliquot was positive) was only improved by approximately 1 order of magnitude. The packed bed process has the potential of a 20-fold improvement in the limit of detection compared to Qiagen plus precipitation, based on a mass balance analysis for the entire DNA concentration and purification processes. Figure ES1 shows a mass balance for all the DNA processing steps. The packed bed process minimizes losses from elution, precipitation, and pipetting (aliquoting and transferring). Figure ES1 assumes that 100 copies of DNA serve as the input sample. Efficiencies for each step have been estimated based on our experiences or a worst case scenario (for example, a 50% loss was assumed for pipetting). Table ES1 summarizes the number of copies that are the input template for PCR assuming 100 copies of DNA are processed through the three options detailed in Figure ES1.Theoretically a 20-fold increase in the number of starting copies in the PCR reaction is gained when the DNA is concentrated, purified and then amplified directly on the surface of the beads in the packed bed.
Date: February 5, 2008
Creator: Wheeler, E K; Erler, A M & Seiler, A
Partner: UNT Libraries Government Documents Department

On-chip real-time single-copy polymerase chain reaction in picoliter droplets

Description: The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10{sup 6} smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of {approx}18, twenty cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.
Date: April 20, 2007
Creator: Beer, N R; Hindson, B; Wheeler, E; Hall, S B; Rose, K A; Kennedy, I et al.
Partner: UNT Libraries Government Documents Department

Field Deployable DNA analyzer

Description: This report details the feasibility of a field deployable DNA analyzer. Steps for swabbing cells from surfaces and extracting DNA in an automatable way are presented. Since enzymatic amplification reactions are highly sensitive to environmental contamination, sample preparation is a crucial step to make an autonomous deployable instrument. We perform sample clean up and concentration in a flow through packed bed. For small initial samples, whole genome amplification is performed in the packed bed resulting in enough product for subsequent PCR amplification. In addition to DNA, which can be used to identify a subject, protein is also left behind, the analysis of which can be used to determine exposure to certain substances, such as radionuclides. Our preparative step for DNA analysis left behind the protein complement as a waste stream; we determined to learn if the proteins themselves could be analyzed in a fieldable device. We successfully developed a two-step lateral flow assay for protein analysis and demonstrate a proof of principle assay.
Date: February 9, 2005
Creator: Wheeler, E; Christian, A; Marion, J; Sorensen, K; Arroyo, E; Vrankovich, G et al.
Partner: UNT Libraries Government Documents Department

Comparison of Packed Beds and Qiagen Columns for Recovering Trace Amounts of B. anthracis DNA from Liquid Suspensions

Description: The goal of this work was to optimize and evaluate LLNL's in-bed amplification technology to improve the level of detection for suspensions containing trace amounts of anthracis DNA. The binding/cleaning performance of the packed bed is compared to the conventional commercial approach; Qiagen column cleanup and elution, followed by detection through an ex-situ amplification process. Five liquid suspensions were spiked with B.anthracis DNA in concentration series. These suspensions were: (1) water, (2) water with EDTA, (3) dirty water from carpet extraction, (4) dirty carpet extraction with phosphate buffered saline (PBS) plus 0.1% Tween 20 plus 0.1% gelatin, and (5) a subway aerosol collected in water. Each suspension matrix was spiked with DNA and injected (in replicate) into either Qiagen Microcolumns (using the kit processing instructions) or LLNL's packed bed (using the LLNL in-bed purification and amplification protocol). The process output was assayed by quantitative polymerase chain reaction (QPCR). Table ES-1 shows the level of DNA (pg per 100 uL of input suspension) that resulted in successful amplification for all reactions (X=Y), and the level for which at least one of the reactions was successful (X>0). For each suspension and DNA concentration, there were Y QPCR assays of which X showed successful amplification. LLNL's packed bed technology outperformed Qiagen Microcolumns for all five suspensions, typically by one order of magnitude in both the limit of assured detection (all reactions positive), and the lower limit of detection (some reactions positive).
Date: June 23, 2006
Creator: Sorensen, K; Arroyo, E; Erler, A; Christian, A T; Camp, D & Wheeler, E K
Partner: UNT Libraries Government Documents Department

On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets

Description: The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment, and will be useful in viral discovery and gene-profiling applications.
Date: December 19, 2007
Creator: Beer, N R; Wheeler, E; Lee-Houghton, L; Watkins, N; Nasarabadi, S; Hebert, N et al.
Partner: UNT Libraries Government Documents Department

Small Sample Whole-Genome Amplification

Description: Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.
Date: September 20, 2005
Creator: Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P et al.
Partner: UNT Libraries Government Documents Department

Hybridization and Selective Release of DNA Microarrays

Description: DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to single spots to release hybridized DNA. This work leverages LLNL expertise in optics, microfluids, and bioinformatics.
Date: November 29, 2011
Creator: Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J et al.
Partner: UNT Libraries Government Documents Department

Flow Through PCR Module of BioBriefcase

Description: The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.
Date: September 19, 2005
Creator: Arroyo, E S; Wheeler, E K; Hindson, B; Nasarabadi, S; Vrankovich, G; Bell, P et al.
Partner: UNT Libraries Government Documents Department

Scatter Loss From Environmental Degradation of KDP Crystals

Description: Severe scattering losses from KDP crystals have been correlated with the exposure of porous sol AR coated crystals to ambient humidity. The scattering is attributed to formation of etch pits which develop under the coating on the KDP surface along crystallographic axes. This high angle scattering can in turn produce laser damage of downstream optics either through modulation of the beam or by optic contamination from ablation of adjacent metal structures. We have developed a simple tool to characterize the evolution of scatter from sol-coated KDP surfaces. We have measured the rate of etch pit formation as a function of relative humidity and surface treatment using both microscopy and scattering. We will discuss various surface treatments which can be utilized to retard or eliminate the environmental degradation of KDP crystals.
Date: December 17, 1999
Creator: Wheeler, E.K.; McWhirter, J.; Whitman, P.K.; Thorsness, C.; De Yoreo, J.; Thomas, I. et al.
Partner: UNT Libraries Government Documents Department

FY10 Engineering Innovations, Research and Technology Report

Description: This report summarizes key research, development, and technology advancements in Lawrence Livermore National Laboratory's Engineering Directorate for FY2010. These efforts exemplify Engineering's nearly 60-year history of developing and applying the technology innovations needed for the Laboratory's national security missions, and embody Engineering's mission to ''Enable program success today and ensure the Laboratory's vitality tomorrow.'' Leading off the report is a section featuring compelling engineering innovations. These innovations range from advanced hydrogen storage that enables clean vehicles, to new nuclear material detection technologies, to a landmine detection system using ultra-wideband ground-penetrating radar. Many have been recognized with R&D Magazine's prestigious R&D 100 Award; all are examples of the forward-looking application of innovative engineering to pressing national problems and challenging customer requirements. Engineering's capability development strategy includes both fundamental research and technology development. Engineering research creates the competencies of the future where discovery-class groundwork is required. Our technology development (or reduction to practice) efforts enable many of the research breakthroughs across the Laboratory to translate from the world of basic research to the national security missions of the Laboratory. This portfolio approach produces new and advanced technological capabilities, and is a unique component of the value proposition of the Lawrence Livermore Laboratory. The balance of the report highlights this work in research and technology, organized into thematic technical areas: Computational Engineering; Micro/Nano-Devices and Structures; Measurement Technologies; Engineering Systems for Knowledge Discovery; and Energy Manipulation. Our investments in these areas serve not only known programmatic requirements of today and tomorrow, but also anticipate the breakthrough engineering innovations that will be needed in the future.
Date: January 11, 2011
Creator: Lane, M A; Aceves, S M; Paulson, C N; Candy, J V; Bennett, C V; Carlisle, K et al.
Partner: UNT Libraries Government Documents Department