Standard reference material 2366 for measurement of human cytomegalovirus DNA.

Human cytomegalovirus (CMV), classified as human herpesvirus 5, is ubiquitous in human populations. Infection generally causes little illness in healthy individuals, but can cause life-threatening disease in those who are immunocompromised or in newborns through complications arising from congenital CMV infection. An important aspect in diagnosis and treatment is to track circulating viral load with molecular methods, particularly with quantitative PCR.

Molecular diagnostics: harmonization through reference materials, documentary standards and proficiency testing.

There is a great need for harmonization in nucleic acid testing for infectious disease and clinical genetics. The proliferation of assay methods, the number of targets for molecular diagnostics and the absence of standard reference materials contribute to variability in test results among laboratories. This article provides a comprehensive overview of reference materials, related documentary standards and proficiency testing programs.

International standards and reference materials for quantitative molecular infectious disease testing.

The utility of quantitative molecular diagnostics for patient management depends on the ability to relate patient results to prior results or to absolute values in clinical practice guidelines. To do this, those results need to be comparable across time and methods, either by producing the same value across methods and test versions or by using reliable and stable conversions. Universally available standards and reference materials specific to quantitative molecular technologies are critical to this process but are few in number.

Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence.

The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA.

Potential primary measurement tool for the quantification of DNA.

An automated sample introduction system, utilizing a demountable direct injection high-efficiency nebulizer (d-DIHEN), is successfully incorporated for the first time with an inductively coupled plasma optical emission spectrometer (ICP-OES) for the measurement of the phosphorus content in acid-digested nucleotides and deoxyribonucleic acid (DNA). With this experimental setup, the solution uptake rate and volume are reduced from 170 to 30 microL min(-1) and from 10 to 2.4 mL, respectively, thereby reducing the required DNA sample mass for solutions containing 3 microg g(-1) P from 300 to 72 microg of DNA, in comparison to previous analyses in our laboratory using a glass concentric nebulizer with cyclonic spray chamber arrangement.

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex.

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.

A study of comparability in amplified fragment length polymorphism profiling using a simple model system.

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated.

Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR.

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality.

Traceable phosphorus measurements by ICP-OES and HPLC for the quantitation of DNA.

Measurement of the phosphorus content of nucleotides and deoxyribonucleic acid (DNA) offers an approach to the quantitation of nucleic acids that is traceable to the SI. Such measurements can be an alternative to the commonly used spectroscopic tools that are not traceable. Phosphorus measurements of thymidine 5'-monophosphate (TMP) and acid-digested plasmid and genomic DNA preparations were made using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES) and high-performance liquid chromatography (HPLC) and compared for bias and uncertainty.

Structural analysis of ligand binding and catalysis in chorismate lyase.

Chorismate lyase (CL) removes the pyruvyl group from chorismate to provide 4-hydroxybenzoate (4HB) for the ubiquinone pathway. We previously reported the crystal structure at 1.4A resolution of the Escherichia coli CL with bound 4HB product, showing that the product is bound in an internal cavity behind two flaps.

An inter-platform repeatability study investigating real-time amplification of plasmid DNA.

Background: The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms - the LightCycler, ABI PRISM 7700 and Rotor Gene 3000.

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV-VIS absorption study.

Putidaredoxin (Pdx) is an 11,400-Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3+/-0.

Structure of C73G putidaredoxin from Pseudomonas putida.

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein.

Crystallization and phasing of alanine dehydrogenase from Archaeoglobus fulgidus.

Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene.

Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA.

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn.

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy.

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine.

Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate.

Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.