Genotyping accuracy of high-resolution DNA melting instruments

pre-printHigh-resolution DNA melting is a closed-tube method for genotyping and variant scanning that depends on the thermal stability of PCR-generated products. Instruments vary in thermal precision, sample format, melting rates, acquisition, and software. Instrument genotyping accuracy has not been assessed. Each genotype of the single nucleotide variant (SNV) (c.3405-29A>T) of CPS1 (carbamoyl-phosphate synthase 1, mitochondrial) was amplified by PCR in the presence of LCGreen Plus with 4 PCR product lengths. After blinding and genotype randomization, samples were melted in 10 instrument configurations under conditions recommended by the manufacturer. For each configuration and PCR product length, we analyzed 32-96 samples (depending on batch size) with both commercial and custom software. We assessed the accuracy of heterozygote detection and homozygote differentiation of a difficult, nearest- neighbor symmetric, class 4 variant with predicted <Tmof 0.00 °C

Dynamic general equilibrium analysis of improved weed management in Australia's winter cropping systems

A recent analysis indicated that the direct financial cost of weeds to Australia’s winter grain sectorwas approximately $A1.2bn in 1998–1999. Costs of thismagnitude represent a large recurring productivity loss in an agricultural sector that is sufficient to impact significantly on regional economies.Using amulti-regional dynamic computable general equilibrium model, we simulate the general equilibrium effects of a hypothetical successful campaign to reduce the economic costs of weeds. We assume that an additional$50m of R&D spread over five years is targeted at reducing the additional costs and reduced yields arising from weeds in various broadacre crops. Following this R&D effort, one-tenth of the losses arising from weeds is temporarily eliminated, with a diminishing benefit in succeeding years. At the national level, there is a welfare increase of $700m in discounted net present value terms. The regions with relatively high concentrations of winter crops experience small temporary macroeconomic gains.CGE modelling, dynamics, weed management, Crop Production/Industries,

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments

pre-printBACKGROUND: Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. METHODS: A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. RESULTS: The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. CONCLUSIONS: Simple DNAextension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations,DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR

Influence of Nitrogen Fertilization on the Quality and Quantity of Streamflow from a Forested Watershed

This project was designed to determine the effects of nitrogen fertilization on the quality and quantity of streamflow eminating from an eastern hardwood forest watershed. A 40.67 ha watershed, located in mountainous eastern Kentucky, was aerially fertilized in late April 1975. The forest stand was principally oak, hickory, and yellow poplar, 50 - 55 years of age and in a relatively undisturbed condition. A helicopter applied anunonium nitrate at a rate of 504 kg/ha. Because a large part of applied nitrogen fertilizer ends up in the highly mobile nitrate nitrogen.form, this is the principal ion monitored in this study. No effort was made to avoid live streams during application and, consequently, very high levels of nitrate nitrogen were detected (640 mg/1) in streamfiow within the watershed. Levels potentially toxic to humans and animals persisted in the streamflow for several days following application. Although elevated concentrations of nitrate nitrogen persisted in streamflow leaving the watershed over a two year period no algal blooms or excessive growth of aquatic plants were noted. Rather high concentrations of nitrate nitrogen were found in the soils of the watershed, with greatest concentrations in the surface layer (0 - 5 cm), intermediate amounts at 15 - 20 cm, and the lowest concentrations at the 41 - 46 cm depth. The effects of the fertilizer application on soils persisted less than one year in the 0 - 46 cm depth sampled. Analysis of streamf1ow records indicated a reduction in water yield the first and second growing seasons after treatment. Gross budgeting of nitrate nitrogen inputs vs. outputs suggests this anion accumulates on these relatively undisturbed watersheds at an annual rate of 3 to 5 kg/ha

A Kerr Polarization Controller

Kerr-effect-induced changes of the polarization state of light are well known in pulsed laser systems. An example is nonlinear polarization rotation, which is critical to the operation of many types of mode-locked lasers. Here, we demonstrate that the Kerr effect in a high-finesse Fabry-Pérot resonator can be utilized to control the polarization of a continuous wave laser. It is shown that a linearly-polarized input field is converted into a left- or right-circularly-polarized field, controlled via the optical power. The observations are explained by Kerr-nonlinearity induced symmetry breaking, which splits the resonance frequencies of degenerate modes with opposite polarization handedness in an otherwise symmetric resonator. The all-optical polarization control is demonstrated at threshold powers down to 7 mW. The physical principle of such Kerr effect-based polarization controllers is generic to high-Q Kerr-nonlinear resonators and could also be implemented in photonic integrated circuits. Beyond polarization control, the spontaneous symmetry breaking of polarization states could be used for polarization filters or highly sensitive polarization sensors when operated close to the symmetry-breaking point

Frequency comb metrology with an optical parametric oscillator

We report on the first demonstration of absolute frequency comb metrology with an optical parametric oscillator (OPO) frequency comb. The synchronously-pumped OPO operated in the 1.5-μm spectral region and was referenced to an H-maser atomic clock. Using different techniques, we thoroughly characterized the frequency noise power spectral density (PSD) of the repetition rate frep, of the carrier-envelope offset frequency fCEO, and of an optical comb line νN. The comb mode optical linewidth at 1557 nm was determined to be ~70 kHz for an observation time of 1 s from the measured frequency noise PSD, and was limited by the stability of the microwave frequency standard available for the stabilization of the comb repetition rate. We achieved a tight lock of the carrier envelope offset frequency with only ~300 mrad residual integrated phase noise, which makes its contribution to the optical linewidth negligible. The OPO comb was used to measure the absolute optical frequency of a near-infrared laser whose second-harmonic component was locked to the F = 2→3 transition of the 87Rb D2 line at 780 nm, leading to a measured transition frequency of νRb = 384,228,115,346 ± 16 kHz. We performed the same measurement with a commercial fiber-laser comb operating in the 1.5-μm region. Both the OPO comb and the commercial fiber comb achieved similar performance. The measurement accuracy was limited by interferometric noise in the fibered setup of the Rb-stabilized laser

Scaling of the Critical Function for the Standard Map: Some Numerical Results

The behavior of the critical function for the breakdown of the homotopically non-trivial invariant (KAM) curves for the standard map, as the rotation number tends to a rational number, is investigated using a version of Greene's residue criterion. The results are compared to the analogous ones for the radius of convergence of the Lindstedt series, in which case rigorous theorems have been proved. The conjectured interpolation of the critical function in terms of the Bryuno function is discussed.Comment: 26 pages, 3 figures, 13 table

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

BACKGROUND: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results