Spectrophotometer for nucleic acid measurements
A spectrophotometer for nucleic acid measurement is an analytical instrument used in molecular biology to determine the average concentrations of the nucleic acids DNA or RNA present in a mixture, as well as their purity. To date, there are two main approaches used by scientists to quantitate DNA or RNA. These are spectrophotometry and fluorescence tagging.
Spectrophotometric analysis
One of the more commonly used practices to quantitate DNA or RNA is the use of spectrophotometric analysis using a spectrophotometer.[1] Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet light in a specific pattern. In the case of DNA and RNA, a sample that is exposed to ultraviolet light at a wavelength of 260 nanometres (nm) will absorb that ultraviolet light. The resulting effect is that less light will strike the photodetector and this will produce a higher optical density (OD)
Calculations
The optical density [2] is generated from equation:
- Optical Density= Log (Intensity of Incident Light / Intensity of
Transmitted Light)
In practical terms, a sample that contains no DNA or RNA should not
absorb any of the ultraviolet light and therefore produce an OD of 0
Optical Density=Log (100/100)=0
When using spectrophotometric analysis to determine the concentration of DNA or RNA, the Beer-Lambert law is used to determine unknown concentrations without the need for standard curves. In essence, the Beer Lambert Law makes it possible to relate the amount of light absorbed to the concentration of the absorbing molecule. The following absorbance units to nucleic acid concentration conversion factors are used to convert OD to concentration of unknown nucleic acid samples:
- A260 dsDNA = 50 µg/ml
- A260 ssDNA = 37 µg/ml
- A260 ssRNA = 40 µg/ml
Conversion factors
When using a 10 mm path length, simply multiply the OD by the conversion factor to determine the concentration. Example, a 2.0 OD dsDNA sample corresponds to a sample with a 100 ug/ml concentration.
When using a path length that is shorter than 10mm, the resultant OD will be reduced by a factor of 10/path length. Using the example above with a 3 mm path length, the OD for the 100 ug/ml sample would be reduced to 0.6. To normalize the concentration to a 10mm equivalent, the following is done: 0.6 OD X (10/3) * 50 ug/ml=100 ug/ml
Most spectrophotometers allow selection of the nucleic acid type and path length such that resultant concentration is normalized to the 10 mm path length which is based on the principles of Beer's law.
A260 as quantity measure
The "A260 unit" is used a quantity measure for nucleic acids. One A260 unit is the amount of nucleic acid contained in 1 mL and producing an OD of 1. The same conversion factors apply, and therefore, in such contexts:
- 1 A260 unit dsDNA = 50 µg
- 1 A260 unit ssDNA = 37 µg
- 1 A260 unit ssRNA = 40 µg
Determination of sample purity
The secondary benefit of using spectrophotometric analysis for nucleic acid quantitation is the ability to determine sample purity using the 260 nm:280 nm calculation. A pure DNA sample will yield an A260/280 of approximately 1.8. For RNA, a pure sample will yield anA260/280 of approximately 2.0. These ratios are commonly used to assess the amount of protein contamination that is left from the nucleic acid isolation process since proteins absorb at 280 nm.
Fluorescence tagging
In some cases, scientists elect to quantitate DNA and RNA using fluorescence tagging methods with dyes that are highly specific to DNA or RNA. The benefit of fluorescence quantitation of DNA and RNA is the improved sensitivity over spectrophotometric analysis. Although, that increase in sensitivity comes at the cost of a higher price per sample and a lengthier sample preparation process. Additionally, there is not currently a fluorescence method to determine protein contamination of a DNA sample that is similar to the 260 nm/280 nm spectrophotometric version.
References
- ↑ Huss, Volker A.R.; Festl, Herbert; Schleifer, Karl Heinz (1983). "Studies on the spectrophotometric determination of DNA hybridization from renaturation rates". Systematic and Applied Microbiology 4 (2): 184–192. doi:10.1016/S0723-2020(83)80048-4. ISSN 0723-2020.
- ↑ IUPAC, Compendium of Chemical Terminology. Online edition: "absorbance".