Specificity direct readout

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Potassium also can be measured by ICP/AES. The wavelengths at which it can be analyzed without interference from other metals are 766.49 and 769.90 nm. Other wavelengths may be used. Potassium ion in aqueous solution can be identified quantitatively by using a potassium ion-selective electrode attached to a pH meter having an expanded millivolt scale or to a specific ion meter having a direct readout concentration scale for potassium. 

Several electrolytic-conductivity detectors are produced (Table 3.5). The Laboratory Data Control Model 701 Conducto Monitor (Fig.3.59) may be operated in either a differential mode or an absolute mode. It provides direct readout in units of specific conductance and differences as small as 0.01% in the differential mode between the carrier and the carrier plus solute can be measured. The dynamic range of linearity is 0.01-100,000 pSl 1 /cm. The detector can function in solvents ranging from distilled water to concentrated salt solutions without the necessity of changing the cell. The volume of the cell is 2.5 pi, and the nominal cell constant is 20 cm-1. This type of detector is of use mainly in high-speed ion-exchange chromatography for the detection of ionic species. 

TBP binds the minor groove of AT rich DNA with a clear sequence preference (the consensus TATA box has the sequence T A T A t/a A t/a X 16)). Understanding the basis for this selectivity is complicated by the fact that AT and TA basepairs are very similar in the minor groove, which precludes direct readout as a means for specific sequence recognition (77). Because TBP distorts the DNA, it is likely that sequence dependent DNA bendability plays an important role in determining the observed sequence specificity 18). 

DNA. By far, the most common secondary structure for DNA is the double helix. Sequence-specific recognition of double-helical DNA relies on several factors. First, the array of fimctional groups exposed in the major and minor grooves can be read by DNA-binding ligands. This is referred to as direct readout. However, more subtle factors may also be involved, such as sequence-dependent variation in the shape, flexibility, and hydration of the DNA. Ligands which discriminate among sequences based on these parameters utilize indirect readout of the sequence information. 

As for future trends, some speculation will be advanced. Some are already on the way all have been given much serious thought. Some form of direct readout is a distinct possibility and will probably simplify the work of interpretation considerably. Interferometric methods to improve resolution in specific regions may be used. 

As previously mentioned, the calibration accuracy specification on high-performance instruments applies to the direct readout element of the monochromator, which is usually a counter or a dial. The large-size-chart abscissa scales generally used with these instruments limit the accuracy on the chart itself more severely due to the humidity sensitivity of even high-grade recorder paper, which affects the dimensional stability. Sprocket-driven strip-chart recorders tend to be self-compensating for this effect. For less versatile instruments, the recorder paper itself, usually with a smaller abscissa scale, is used as the abscissa reference. 

ALIS reports only compounds that bind directly to the target of interest, preventing false positives that arise from off-target activity or interactions with substrates or other reagents. Since ALIS directly identifies bound components by MS, the incidence of false positives based on bulk effects and non-specific binding is lower than that of biochemical assays that yield a secondary readout of activity. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

Many of these approaches rely on the differential hybridization of target DNAs that are perfectly matched with probe sequences to achieve highly specific and accurate target selection. Other assays use the sensitivity of DNA-mediated charge transport to duplex structure in order to signal the presence of a sequence of interest. Efforts also have been directed toward exploiting non-faradaic processes unique to DNA-modified surfaces as the basis for electrochemical readout. This chapter discusses each of these methods, and is intended to highlight how the DNA/electrode interface can... 

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