M. B. Nelson

A quantitative model of error accumulation during PCR amplification

The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process.

Target diagnostic system for the national ignition facility (invited)

A review of recent progress on the design of a diagnostic system proposed for ignition target experiments on the National Ignition Facility (NIF) will be presented. This diagnostic package contains an extensive suite of optical, x ray, gamma ray, and neutron diagnostics that enable measurements of the performance of both direct and indirect driven NIF targets. The philosophy used in designing all of the diagnostics in the set has emphasized redundant and independent measurement of fundamental physical quantities relevant to the operation of the NIF target. A unique feature of these diagnostics is that they are being designed to be capable of operating in the high radiation, electromagnetic pulse, and debris backgrounds expected on the NIF facility. The diagnostic system proposed can be categorized into three broad areas: laser characterization, hohlraum characterization, and capsule performance diagnostics. The operating principles of a representative instrument from each class of diagnostic employed in t...

LaNSA: A large neutron scintillator array for neutron spectroscopy at Nova

A very sensitive neutron time‐of‐flight spectrometer is now in use at Nova. LaNSA consists of 960 channels of a neutron sensitive liquid scintillator (10×10×10 cm) coupled to a photomultiplier tube followed by a discriminator, TDC, and ADC to allow the measurement of neutron arrival time as well as pulse size. LaNSA is capable of measuring yields as low as 2.3×105 DT neutrons (100 detected hits) with resolution of 2.3 ns (170 keV for 14‐MeV neutrons with 20‐m flight path). Shielding and collimation provide background levels low enough to allow measurement of secondary and tertiary reaction neutrons. Details of design, testing, calibration, and experimental results will be presented.

Neutron spectroscopy with a large neutron time‐of‐flight detector array (LaNSA) (invited)

The recent completion of the Large Neutron Scintillator Array (LaNSA) at the Nova Laser Fusion Facility has allowed low yield neutron energy spectra to be measured for ICF implosions. These spectra can be used to determine some fundamental implosion characteristics such as fuel areal density and ion temperature. Details of how neutron energy spectra are measured with an array of single particle detectors will be presented, as well as experimental data showing the application of these spectra to the determination of implosion parameters. Particular emphasis will be placed on measurements of secondary DT neutrons produced from initially pure deuterium fuel. Determination of the interacting triton energy spectrum from secondary neutron energy spectroscopy and its application to studies of fuel‐pusher mixing will also be discussed.

Neutron yield measurements with current‐mode neutron time‐of‐flight detectors (abstract)

Current‐mode neutron time‐of‐flight detectors (e.g., a scintillator and photomultiplier tube) are frequently used as sensitive detectors for laser fusion neutron yield measurements. For very low yields, as in the measurement of secondary (dt) neutrons from deuterium targets, some subtle effects involving limited statistical samples and the broad ‘‘pulse‐height distribution’’ of a neutron sensitive scintillator arise. These effects are discussed and error treatments are presented. A technique using a pulsed neutron generator is shown to provide, experimentally, all the information required for characterization of a detector. Comparisons to yield measurements made at Nova will be presented. This work was performed under the auspices of the U.S. DOE by the Lawrence Livermore National Laboratory under Contract No. W‐7405‐ENG‐48.

Ranque–Hilsch vortex tube thermocycler for fast DNA amplification and real-time optical detection

An innovative polymerase chain reaction (PCR) thermocycler capable of performing real-time optical detection is described below. This device utilizes the Ranque–Hilsch vortex tube in a system to efficiently and rapidly cycle three 20 μL samples between the denaturation, annealing, and elongation temperatures. The reaction progress is displayed real-time by measuring the size of a fluorescent signal emitted by SYBR green/double-stranded DNA complexes. This device can produce significant reaction yields with very small amounts of initial DNA, for example, it can amplify 0.25 fg (∼5 copies) of a 96 bp bacteriophage λ-DNA fragment 2.7×1011-fold by performing 45 cycles in less than 12 min. The optical threshold (150% of the baseline intensity) was passed 8 min into the reaction at cycle 34. Besides direct applications, the speed and sensitivity of this device enables it to be used as a scientific instrument for basic studies such as PCR assembly and polymerase kinetics.

A technique for shell compression measurements of laser fusion targets by neutron activation of a rubidium tracer

At the Nova Laser, the activation of a rubidium tracer incorporated in the shell of ICF targets has become a standard diagnostic technique for measuring the compressed shell areal density {l angle}{rho}{Delta}R{r angle}. The isotope {sup 85}Rb is activated by 14 MeV implosion neutrons to produce the isomer {sup 84m}Rb(t{sub {1/2}} = 20.5 min) which is used to determine the shell {l angle}{rho}{Delta}R{r angle} while the radioactive isotope {sup 86}Rb(t{sub 1/2} = 18.8 d) is used to determine the fraction of target debris collected as well as to assay the amount of rubidium in the target. The {sup 85}Rb(n,2n){sup 84m}Rb cross-section at 14.1 MeV was measured ({sigma} = 0.514 {plus minus} 0.080 b). Details of the detector system and experimental technique are given. 12 refs., 2 tabs.

Ranque-hilsch vortex tube thermocycler for DNA amplification

Abstract An innovative polymerase chain reaction (PCR) thermocycler using pressurized gas through a Ranque–Hilsch vortex tube is described below. This device can amplify 10 pg of 186 bp Escherichia coli uidA amplicon in a 20 µL sample 3.3 × 108‐fold, by performing 35 cycles in less than 8 min. This PCR amplification corresponds to an overall efficiency of 75%.

Genotyping of DNA using sequence-specific methyltransferases followed by immunochemical detection.

Abstract Modern molecular genetics relies on the ability to map the positions of genes on chromosomes, relative to known DNA markers. The first such DNA markers described were Restriction Fragment Length Polymorphisms, but any restriction endonuclease used for RFLP mapping is just one member of a restriction-modification pair. For each restriction endonuclease, there is a companion methyltransferase (MTase) that has the same DNA sequence specificity. Therefore, in principle, it should be possible to use MTases rather than restriction enzymes to detect polymorphic sites in DNA. We have used sequence-specific DNA MTases to detect genetic polymorphisms in closely related viral pathogens. If at least one MTase recognition site is present in PCR-amplified DNA, then methyl groups are incorporated; if no MTase site is present, then methyl groups are not incorporated. When several different sequence-specific DNA MTase reactions are carried out, the pattern of methyl incorporation defines a DNA MTase genotype. DNA MTase Genotyping (DMG) can be used to rapidly diagnose heritable or infectious diseases, to immunochemically detect DNA at defined 2 to 8 base pair sites, or to characterize the amplicons by constructing ordered maps.

Thermal analysis of the vortex tube based thermocycler for fast DNA amplification: Experimental and two-dimensional numerical results

In this article, experimental and numerical analyses to investigate the thermal control of an innovative vortex tube based polymerase chain reaction (VT-PCR) thermocycler are described. VT-PCR is capable of rapid DNA amplification and real-time optical detection. The device rapidly cycles six 20μl 96bp λ-DNA samples between the PCR stages (denaturation, annealing, and elongation) for 30cycles in approximately 6min. Two-dimensional numerical simulations have been carried out using computational fluid dynamics (CFD) software FLUENT v.6.2.16. Experiments and CFD simulations have been carried out to measure/predict the temperature variation between the samples and within each sample. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the temperature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation a...