The hyperchromic effect is the increase in UV absorption seen in double-stranded DNA upon denaturation of the DNA strand. The effect is also seen in single-stranded DNA with significant intrastrand (secondary structure) interactions upon the disruption of these electrostatic interactions. The effect can be seen by either heating the DNA, altering the pH, or adding a nuclease. We have developed a laboratory experiment based on the exonuclease-induced hyperchromicity of DNA. This experiment is primarily suitable for undergraduate biochemistry courses and has been implemented into our Biochemistry I laboratory curriculum; student results from this laboratory will be presented in this poster. The maximum wavelength (λ<sub>max</sub>) and baseline absorbance signal of both completely complementary double-stranded DNA and single-stranded DNA that is predicted to have significant secondary structure were first determined using a standard UV-vis spectrophotometer. The absorbance at the λ<sub>max</sub> was then followed when either T7 or exonuclease I was introduced to analyze the hyperchromic effect in each system. Upon the introduction of the nuclease there was an obvious, permanent increase in absorbance due to the hyperchromic effect. Aspects such as concentration of enzyme or DNA in each experiment were altered to find conditions suitable for the laboratory setting; an increase in absorbance from 10-100% was seen in our experiments depending on the conditions used. The results can be tied in to discussions involving when accurate concentration measurements are important in research settings, such as in protein-DNA binding assays or PCR. We have included online bioinformatics tools that allow students to predict secondary structure and stability of the single-stranded DNA sequence to determine it may show evidence of the hyperchromic effect. In this poster, we also suggest other options for educators, including single-stranded DNA without a stable secondary structure.

Using UV spectroscopy to follow exonuclease-induced hyperchromicity in the undergraduate biochemistry lab

251st National Meeting (2016)

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