Nucleic acid quantification

Quantifation of nucleic acid after successful isolation is a crucial step impacting all downstream applications. Two of the commonly used methods, spectrophotometry and fluorescence spectroscopy, will be discussed in the following paragraphs.

Spectrophotometry

Spectrophotometry is the commonly used cheap and straightforward method to quantify nucleic acids. However, this method can be easily affected by excess or low amounts of nucleic acids or presence of contaminations.

Defined extinction coefficients for DNA and RNA in absence of a standard curve and a cuvette thickness layer of 10mm are shown below. A dsDNA sample with an absorbance of 1 at 260nm will have a concentration of 50ng/µl.

Extinction coefficient of DNA and RNA

Although the absorbance at 260nm is specific for nucleic acids, contaminations from proteins or solvents can influence the purity. A pure sample will show a ratio of approximately 1.8 for DNA and ~2.0 for RNA at A260/A280. The higher quotient calculated for RNA is due to presence of uracil. Estimated ratios at A260/A230 are between 2.0 and 2.5.

As a rule of thumb the A260/280 ratio is used to determine contaminations of organic origin whereas the A260/230 is used to identity contaminations of ethanol or phenol.

Benefits and drawbacks of spectrophotometry

Spectrophotometry is based on the law by Lambert-Beer. According to this the absorbance of a substance is proportional to its concentration. For nucleic acids the maximum absorbance at 260nm is based on the nucleotides (i.e adenine, thymine/uracil, guanine, cytosine) interacting with the UV light at 260nm.

Flourescence spectroscopy

Flourescence spectroscopy is based on the detection of emitted light from a fluorophore. Fluorophores respond specifically to light in contrast to other molecules. When energy i.e. light hits a fluorophore it puts the whole molecule into an excitation state. The excitation state is known as light absorption. From this excitation state the molecule decays and goes to a lower excitation state. This decay is known as emission. During the decay process energy in form of heat is released allowing the fall back into the relaxed state. Emitted and excited flourophor can be distinguished based on their wavelength. Due to less energy present in the emitted flourophor its wavelength is longer compared to the excited flourophor. This difference in wavelength can be detected using a fluorometer.

Quantification using flourescent dyes

Using flourescence dyes for your samples can be an advantage as the dye can selectivly bind to DNA or RNA. Taken the advantages of flourophores DNA and RNA can be detected and distringued in one sample. Low sample amounts can be measured using flourometric quantification.

Benefits and drawbacks of flourometric measurement

Summary

Flourescence spectroscopy is a very selective and sensitive method however also cost intense.