The first photochemical crosslinking of a protein to a nucleic acid using laser excitation is reported. A single, 120 mJ, 20 ns pulse at 248 nm crosslinks about 10% of bound E. coli RNA polymerase to T7 DNA under the conditions studied. The crosslinking yield depends on mercaptoethanol concentration, and is a linear function of laser intensity. The protein subunits crosslinked to DNA are beta, beta' and sigma.

BackgroundThe performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats.ResultsA low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface.ConclusionsThese results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves.

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Appropriate statistical analysis of the validation data for diagnostic tests facilitates the evaluation of the performance criteria and increases the confidence

SYNOPSISIt is now theoretically possible to obtain absolute accurate values of number-average molecular weight of complex polymers (e.g., branched polymers or copolymers) using size exclusion chromatography (SEC) with only a detector that measures the difference between the eluting polymer solution viscosity and the viscosity of the pure mobile phase ( a differential viscometer [ DV] detector). However, both precision and accuracy of these "DV Mn" values are of concern. In this work, the precision of NBS 706 polystyrene was found to be two to three times worse for the DV Mn than for the conventionally calculated Mn. Also, regarding accuracy, the DV Mn values were affected by the location of the universal calibration curve along the retention volume axis ( a problem intimately associated with the problem of specifying the correct interdetector volume), the sensitivity of the DV detector to low molecular weights present in the sample, and axial dispersion. Each of these sources of error are examined in turn and two methods of calculating M n values are proposed.