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

Abstract

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 λ max 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.

Effect of pendant stereostructure on backbone conformation and enantioseparation ability of helical polyacetylene‐based chiral stationary phases

“…The weakest Cotton effects of S‐pRac might mean that the racemization of site II exerted a more negative influence on the regularity of polymer backbone. The hypochromic effect of S‐pRac at around 340 nm might be attributed to the irregular conformation, similar to that observed in some DNA 43 . Among three PAs with ( S )‐stereocenter in site II, Rac‐pS showed the weakest Cotton effects, indicating that the racemic site I drastically destroyed the stereoregular structure (Figure S25A).…”

“…The hypochromic effect of S-pRac at around 340 nm might be attributed to the irregular conformation, similar to that observed in some DNA. 43 Among three PAs with (S)-stereocenter in site II, Rac-pS showed the weakest Cotton effects, indicating that the racemic site I drastically destroyed the stereoregular structure (Figure S25A). And such irregularity could break the conjugated structure, which was manifested as the absorption blue-shift towards 329 nm.…”

Control of Higher-Order Structures of Platinum-Containing Conjugated Polymers by Ligand Exchange Reactions: Chirality Transfer from Optically Active Ligands to Optically Inactive Polymers

“…The ε of 4 and L2 at about 400 nm did not change, while the ε of 5 and L2 at about 370 nm gradually decreased. It is considered that the resonance of conjugated moieties of the product from 5 and L2 becomes limited by helix formation, in a fashion similar to the hypochromicity of a denaturated single strand random coil DNA folding into a double-strand helix, 31,32 in which stacking and hydrogen bonding interaction occur between the complementary bases. The ligand exchange reaction of 4 with L2 in CH 2 Cl 2 at 0.1 mM was also monitored by DLS measurement.…”

Afm Visualization of Phytoplasma Dna Immobilization and Hybridization Event on Chitosan Modified Spce Surface

“…Observation in all cases of the phytoplasma DNA from SCWL, BGWL and SP similarly and consistently presented the duplex dsDNA and ssDNA profiles as well (data not shown). In general, differentiation in UV light absorption measurement is the only technique for notifying the separation of the DNA strands [14]. The attained AFM image could thus be considered as the first direct visual evidence revealing a DNA separating posture simultaneously after the common denaturation process by heating.…”