'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses.

A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods

Reinvestigation of the Multiepoch Direct Detections of HD 88133 b and Upsilon Andromedae b

We reanalyze the multiepoch direct detections of HD 88133 b and ups And b that were published in Piskorz et al. (2016) and Piskorz et al. (2017), respectively. Using simulations to attempt to reproduce the detections, we find that with the six and seven L-band Keck/NIRSPEC epochs analyzed in the original works, the planets would not have been detectable unless they had unreasonably large radii. HD88133 and ups And both have fairly large stellar radii, which contributed to the difficulty in detecting the planets. We take this opportunity to consider how these planets may have been detectable with the small number of epochs originally presented by running simulations both with the upgraded NIRSPEC instrument and with near-zero primary velocities, as recommended by Buzard et al. (2021). While seven L-band NIRSPEC2.0 epochs with near-zero primary velocities would have allowed a strong (10.8σ) detection of ups And b, many more than six L-band epochs would have been required for a strong detection of HD88133b, which could be due in part to both this system's large stellar radius and low stellar temperature. This work stresses the importance of careful analytic procedures and the usefulness of simulations in understanding the expected sensitivity of high-resolution spectroscopic data. © 2021. The Author(s). Published by the American Astronomical Society..Immediate accessThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]