Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology.
The CheA protein of Escherichia coli is a histidine autokinase that donates its phosphate groups to two target proteins, CheY and CheB, to regulate flagellar rotation and sensory adaptation during chemotactic responses. The amino-terminal third of CheA contains the autophosphorylation site, determinants needed to interact with the catalytic center of the molecule, and determinants needed for specific recognition of its phosphorylation targets. To understand the structural basis for these activities, we examined the domain organization of the CheA phosphotransfer region by using DNA sequence analysis, limited proteolytic digestion, and a genetic technique called domain liberation. Comparison of the functionally interchangeable CheA proteins of E. coli and Salmonea typhimurium revealed two extensively mismatched segments within the phosphotransfer region, 22 and 25 aa long, with sequences characteristic of domain linkers. Both segments were readily susceptible to proteases, implying that they have an extended, flexible structure. In contrast, the intervening segments of the phosphotransfer region, designated P1 and P2 (roughly 140 and 65 aa, respectively), were relatively insensitive, suggesting they correspond to more compactly folded structural domains. Their functional properties were explored by identifying portions of the cheA coding region capable of interfering with chemotactic behavior when "liberated" and expressed as polypeptides. P1 fragments were not inhibitory, but P2 fragments blocked the interaction of CheY with the rotational switch at the flagellar motor, leading to incessant forward swimming. These results suggest that P2 contains CheY-binding determinants which are normally responsible for phosphotransfer specificity. Domain-liberation approaches should prove generally useful for analyzing multidomain proteins and their interaction targets.The chemotaxis machinery of Escherichia coli and Salmonella typhimurium has afforded substantial insight into the information-processing strategies and molecular workings of protein-based signaling circuits (1). These bacteria continuously monitor their chemical environment as they swim about, moving toward beneficial compounds and away from harmful ones. Chemoeffector gradients elicit appropriate swimming responses by changing the cell's pattern of flagellar rotation. Counterclockwise (CCW) rotation produces forward swimming; clockwise (CW) rotation initiates tumbling motions and random directional changes. Most attractants and repellents are detected by a family of transmembrane receptors known as methyl-accepting chemotaxis proteins (MCPs), which communicate with rotational switches at the flagellar motors through a network of cytoplasmic signaling proteins. As in higher organisms, intracellular signaling by bacterial MCPs involves protein phosphorylation and dephosphorylation reactions.
The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization ofB. burgdorferi quantities to the mouse nidogengene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.
CheA is the histidine autokinase in the Escherichia coli chemotaxis signal transduction pathway responsible for coupling of signals received by transmembrane receptors to the response regulators CheY and CheB. Here NMR spectroscopy is used to study a 14 kDa fragment of CheA, residues 124-257, that binds the response regulator CheY. Backbone atom resonance assignments were obtained by analysis of 3D HNCACB, 3D CBCA(CO)NH, and HNCO spectra, whereas side-chain assignments were obtained primarily by analysis of 3D H(CCO)NH, 3D C(CO)NH, 3D HCCH-TOCSY, and 3D 1H, 15N TOCSY-HSMQC spectra. NOE cross peak patterns and intensities as well as torsion angle restraints were used to determine the secondary structure, and a low-resolution structure was calculated by hybrid distance-geometry simulated annealing methods. The CheA124-257 fragment consists of four antiparallel beta strands and two helices, arranged in an "open-faced beta-sandwich" motif, as well as two unstructured ends that correspond to domain linkers in the full-length protein. The 15N-1H correlation spectrum of 15N-labeled CheA124-257 bound to unlabeled CheY shows specific localized changes that may correspond to a CheY-binding face on CheA.
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