Electrophoretic separation of oligonucleotides in denaturing polyacrylamide gels is primarily a function of length-dependent mobility. The 3' terminal nucleotide sequence of the oligonucleotide is a significant, secondary determinant of mobility and separation. Oligomers with 3'-ddT migrate more slowly than expected on the basis of length alone, and thus are better separated from the preceding, shorter oligomers in the sequencing ladder. Oligomers with 3'-ddC are relatively faster than expected, and are therefore less separated. At the 3' penultimate position, -dC- increases and -dT- reduces separation. Purines at the 3' terminal or penultimate positions of oligonucleotides affect separation less than the pyrimidines. These results suggest specific interactions among neighboring nucleotides with important effects on the conformation of oligonucleotides during electrophoresis. These interactions are compared to compression artifacts, which represent more extreme anomalies of length-dependent separation of oligonucleotides. Knowledge of base-specific effects on electrophoretic behavior of DNA oligomers supplements the usual information available for determination of sequences; additionally it provides an avenue to thermodynamic and hydrodynamic investigations of DNA structure.
Spontaneous vibrational Raman scattering (VRS) is produced by a broadband excimer laser at 248 nm (KrF) in a H(2)-air flame and VRS spectra are recorded for lean, stoichiometric, and rich flames. Except at very lean flame conditions, laser-induced fluorescence (LIF) processes interfere with VRS Stokes lines from H(2), H(2)O, and O(2). No interference is found for the N(2) Stokes and N(2) anti-Stokes lines. In a stoichiometric H(2)/air flame, single-pulse measurements of N(2) concentration and temperature (by the VRS Stokes to anti-Stokes ratio) have relative standard deviation of 7.7 and 10%, respectively. These single pulse measurement errors compare well with photon statistics calculations using measured Raman cross sections.
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