The exponentially modified Gaussian (EMG) peak shape is widely used for peak approximation in chromatography. We constructed the EMG peak deconvolution routine for chromatography using a combination of two EMG formulas and linear optimization methods. A convenient way to determine the position of the EMG peak top was found. The routine accounts for the maximum linear range of the detector and can work with out-of-range peaks, where the detector saturation cuts off the top of the peak.The optimization routine is applied to the reconstruction of out-of-range peaks using correctly measured points, so that an analyst can get an idea of the height, area and concentration of such peaks as well as the prediction error in similar cases. Peak reconstruction helps in reducing the number of chromatographic runs during method development and routine work. The possibility of reconstructing out-of-range peaks using the pre-defined peak shape obtained while calibrating is also discussed.
The fine structure of the melting curve for the linear ColB1 DNA ha been obtained. To In the present study we fixed the melted regions with glyoxal to determine the ColEB1 regions whose melting corresponded to peaks in the fine structure of the melting curve.This method h"a earlier been used for localizing the AT-rich regions in T7 DNA revealed by the fine structure of the melting curves /12/. Glyoxal reacts with the amino-and iminogroups of bases and prevents complementary pairing; the product of its reaction with guanine is highly stable /13/.Glyoxal was used to fix the denatured regions appearing in DNA molecules at pre-chosen temperatures within the melting range. The set of denaturation maps subsequently constructed from electron micrographs made it possible to determine the size and location of the DNA regions corresponding to peaks in the fine structure of the melting curve. A combination of spectrophotometry and electron-microscopic visualization of the partially denatured molecules reveals the complete picture of the heat denaturation process. Using these results we construct'ed a map of the distribution of averaged GC-pair content over the ColEl DNA molecule.
The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.
A previously elaborated technique for fixing a chosen partially melted state of DNA with glyoxal was used in a study of the melting process of the replicative form (RF III) of phi X174 DNA. Electron-microscopic maps corresponding to five points of the melting curve of RF III were obtained and compared with the theoretical melting maps obtained in (4) and (6). This comparison clearly shows that only rigorous calculations (4) and not the ones proposed by Azbel (6,7) correctly predict the course of RF III melting.
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