Analyzing Exonuclease-Induced Hyperchromicity by UV Spectroscopy: An Undergraduate Biochemistry Laboratory Experiment

• Megan M. Ackerman ^[1] ; Christopher Ricciardi ^[1] ; David Weiss ^[1] ; Alan Chant ^[2] ; Christina M. Kraemer-Chant ^[1]
  1. [1] Saint Michael's College

     Saint Michael's College

     Town of Colchester, Estados Unidos

     
  2. [2] University of Vermont

     University of Vermont

     City of Burlington, Estados Unidos

     
• Localización: Journal of chemical education, ISSN 0021-9584, Vol. 93, Nº 12, 2016, págs. 2089-2095
• Idioma: inglés
• Texto completo no disponible (Saber más ...)
• Resumen

  • An undergraduate biochemistry laboratory experiment is described that utilizes free online bioinformatics tools along with readily available exonucleases to study the effects of base stacking and hydrogen bonding on the UV absorbance of DNA samples. UV absorbance of double-stranded DNA at the λmax is decreased when the DNA bases are involved in hydrogen bonding and formation of secondary structure. When an exonuclease is added to the solution containing the DNA, the strand is digested and the interactions disappear, leading to an increase in the absorbance called hyperchromicity. This experiment utilizes exonuclease digestion of DNA to show students how base interactions and secondary structure can alter the spectroscopic properties of a sample and, by extension, how apparent concentration as calculated with Beer–Lambert’s law is not necessarily representative of the true concentration of DNA in solution. Teaching applications of this laboratory experiment include enzyme kinetics and activity, secondary structure of single-stranded DNA (and its parallel to RNA structure), and an introduction to bioinformatics.