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The principle underlying the use of sloppy molecular beacon probes in screening assays is that the Tm value of the probe-target hybrid reflects the degree to which the probe sequence is complementary to the target sequence in the amplicon. Although the Tm value of a hybrid formed by a sloppy molecular beacon probe does not provide sufficient information to identify the species from which the amplicon was generated, the simultaneous use of a set of sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, provides a set of Tm values that serves as a unique, species-specific signature.
Determination of the stability of probe-target hybrids formed between sloppy molecular beacon probe A and seven different mycobacterial target oligonucleotides. (A) The fluorescence intensity of the molecular beacon in each hybrid was measured as a function of temperature. A drop in fluorescence intensity occurs at those temperatures where each hybrid dissociates. The increase in fluorescence intensity observed at higher temperatures with each species, and with the no-target control (black line), is due to the melting apart of the probe's hairpin stem. Each of the seven target oligonucleotides formed hybrids of differing stability (from weakest to strongest: M. aichiense, M. diernhoferi, M. abscessus, M. lentiflavum, M. senegalense, M. terrae, and M. asiaticum). (B) Utilizing the data from the left-hand panel, the derivative of fluorescence intensity with respect to temperature (dI/dT) is plotted as a function of temperature. (C) The data within approximately 2°C of each peak in the middle panel were normalized to the same height.
Dependence of hybrid stability on the relatedness of the probe sequence to the target sequence. The Tm of the hybrids formed by each of the four sloppy molecular beacon probes with 27 different species-specific oligonucleotide targets is plotted as a function of the number of mismatched base pairs in the hybrid. In general, the more mismatches present, the lower the Tm. Since each of the four molecular beacons possessed a different probe sequence, the set of four Tm values obtained for a particular species-specific target serves as a unique signature that identifies the species that is present. Results for M. chubuense (red), M. malmoense (blue), and M. triviale (orange) provide examples of unique species-specific signatures.
Determination of the stability of hybrids formed by sloppy molecular beacon probe A with seven different mycobacterial target amplicons. The fluorescence intensity of the molecular beacon in each hybrid is plotted as a function of temperature. A drop in fluorescence intensity is observed at those temperatures where each hybrid dissociates. The black line shows the fluorescence intensity of probe A in the no-target control reaction mixture. The hybrids formed from these seven mycobacterial amplicons gave the following Tm values: M. aichiense, 48°C; M. rhodesiae, 54°C; M. abscessus, 57°C; M. lentiflavum, 60°C; M. szulgai, 62°C; M. avium, 65°C; and M. terrae, 67°C.
Species-specific signatures for 27 different mycobacteria. Each PCR assay was initiated with genomic DNA from a different mycobacterial species. The normalized derivatives of the fluorescence intensity of the hybrids formed by the binding of the resulting amplicons to the differently colored sloppy molecular beacon probes present in each reaction mixture (dI/dT) are plotted as a function of temperature. The set of three or four Tm values determined in each reaction by melting apart these hybrids serves as a unique signature that identifies the mycobacterial species whose genomic DNA was used to initiate amplification.
Schematic representation of the four sloppy molecular beacon probes. Each probe possessed a different sequence and a differently colored fluorophore, enabling the four probes to be used simultaneously in the same reaction mixture and to be distinguished from each other by the spectrofluorometric thermal cycler used to perform the assays. Probe A was labeled with fluorescein (red) and dabcyl (gray), probe B was labeled with Alexa Fluor 546 (blue) and dabcyl, probe C was labeled with Alexa Fluor 594 (orange) and BHQ2 (black), and probe D was labeled with Cy5 (green) and BHQ2.
It is common to think of fluorescent hybridization probes as tools to determine whether a particular target sequence is present or absent in a sample by observing whether a fluorescence signal occurs or does not occur after incubation of the sample with the probe. However, these results demonstrate that sloppy molecular beacon probes form a fluorescent hybrid with many different target sequences even though they are not perfectly complementary to the targets.
In general, it is desirable to utilize smaller amplicons, if possible, as this is likely to limit the formation of secondary and tertiary structures in the target strand that restrict the access of the probes. It may also be worthwhile to explore the use of sloppy molecular beacon probes in assay formats that generate single-stranded RNA amplicons (8) rather than DNA amplicons, such as assays that employ transcription-mediated amplification or nucleic acid sequence-based amplification. These target amplification techniques do not generate cRNA strands. Moreover, they might be more sensitive because they can directly amplify a segment of the abundant 16S rRNA rather than a segment of the 16S rRNA gene, which is present only in one or two copies per cell.
The results of the experiments reported here provide guidance as to how sloppy molecular beacon probes should best be designed. In order to ensure that the magnitude of the fluorescence signal generated by the probe-target hybrids is sufficiently intense to enable their Tm values to be determined, the probe sequence of each molecular beacon should be selected in such a manner that all of the species-specific hybrids will contain less than 10 mismatched base pairs (and have Tm values above 45°C), assuming that the target sequence is about 40 nucleotides in length. In addition, in order to ensure a diverse set of species-specific signatures, the sequence of each of the probes should differ from the sequence of each of the other probes by at least four nucleotide substitutions. It would be helpful to have a computer program for the selection of an optimal set of probe sequences. However, the selection of a useful set of probe sequences is not a difficult task, since we obtained good results by simply examining the set of target sequences and designing probes that varied from one another in those regions of the target sequences that contained the most sequence variations.
Although these screening assays are designed to detect the presence of a single species in a sample, the melting curves that are obtained will often enable the identification of two different species when they are both present. In these situations, there will usually be two separate drops in fluorescence intensity in the melting curve for each of the sloppy molecular beacon probes, with each drop occurring at a Tm value characteristic of one of the two species.
Real-world screening assays can be designed to identify species that occur in different genera and to differentiate species under conditions where the identification of the species that is present in a sample has clinical significance. In this regard, we have been developing an assay that utilizes sloppy molecular beacon probes to identify sepsis-causing bacteria of differing genera in normally sterile human blood samples (S. Chakravorty et al., unpublished data).
Recent reports suggest that there has been an increase in the number of retractions and corrections of published articles due to post-publication detection of problematic data. Moreover, fraudulent data and sloppy science have long-term effects on the scientific literature and subsequent projects based on false and unreproducible claims. At the JCI, we have introduced several data screening checks for manuscripts prior to acceptance in an attempt to reduce the number of post-publication corrections and retractions, with the ultimate goal of increasing confidence in the papers we publish.
When I was growing up, Manwich, the canned sloppy joe sauce, was a family favorite for making and enjoying quick skillet meals. My still-speedy take uses gochujang for a smoky, spicy undertone. Treat this recipe as a way to hone your ideal levels of heat and sweetness by playing with the amount of gochujang, ketchup, and vinegar. Potato buns provide an ideal soft landing and cold pickle spears are a bright, crunchy counterpoint. When it comes to sloppy joes, the sloppier the better, so be sure to load up those buns and arm yourself with plenty of napkins!
In this work, we begin by empirically testing 17 systems biology models from the literature, examining the sensitivity of their behavior to parameter changes. Strikingly, we find that Brown et al.'s model is not unique in its sloppiness; every model we examine exhibits a sloppy parameter sensitivity spectrum. (Thus, in the models we've examined, sloppiness is also universal in the common English sense of ubiquity.) We then study the implications of sloppiness for constraining parameters and predictions. We argue that obtaining precise parameter values from collective fits will remain difficult even with extensive time-series data, because the behavior of a sloppy model is very insensitive to many parameter combinations. We also argue that, to usefully constrain model predictions, direct parameter measurements must be both very precise and complete, because sloppy models are also conversely very sensitive to some parameter combinations. Tests over our collection of models support the first prediction, and detailed analysis of the model of Brown et al. supports the second contention.
Naively, one might expect the stiff eigenvectors to embody the most important parameters and the sloppy directions to embody parameter correlations that might suggest removable degrees of freedom, simplifying the model. Empirically, we have found that the eigenvectors often tend to involve significant components of many different parameters; plots of the four stiffest eigenvectors for each model are in Text S1. This is understandable theoretically; the nearly degenerate sloppy eigenvectors should mix, and the stiff eigenvectors can include arbitrary admixtures of unimportant directions to a given important parameter combination. (Indeed, in analogous random-matrix theories, the eigenvectors are known to be uncorrelated random vectors .) While the relatively random eigenvectors studied here may not be useful in guiding model reduction, more direct explorations of parameter correlations have yielded interesting correlated parameter clusters . 781b155fdc