by Marika Raitio
The Nobel Laureate, and famous biochemist, Bruce Merrifield was once quoted as saying that the spectrophotometer was "probably the most important instrument ever developed towards the advancement of bioscience" [1]. Yet this workhorse of the laboratory, frequently used to calculate DNA and RNA concentrations, can be limited in its application as sample sizes are often small and the sample required for quantitation can further reduce the volume remaining for downstream experimentation. The µDrop Plate has therefore been developed to extend the range of nucleic acid concentration measurements from small volumes without the need for any dilutions.
The spectrophotometer works on the premise that analyte molecules will absorb photons of light that are passed through it. By measuring the difference between the light intensity emitted by the spectrophotometer and the intensity of light detected following passage through the sample, it is possible to reliably and accurately calculate analyte concentrations. The use of the spectrophotometer spans a wide range of scientific fields, from physics to material science and even forensic science. In the field of molecular biology, the spectrophotometer is one of the most widely used instruments and its most common applications include measuring enzymatic reactions and the quantification of DNA prior to digestion by restriction enzymes or PCR amplification.
UV photometry: shedding light on DNA analysis
DNA molecules absorb wavelengths of light in the ultra-violet (UV) range, hence UV photometry is one of the most frequently used methods to measure nucleic acid concentrations. One of the major benefits of the technique is that it is non-destructive, meaning that samples can be used for further analysis.
As UV light is passed through a sample containing DNA, the aromatic rings on the bases absorb the light. A maximum absorption occurs at the 260nm wavelength. Double-stranded and single-stranded DNA absorb light differently due to their structural configurations: in double-stranded DNA the bases tend to be stacked on top of each other and are shielded from the light, therefore less light will be absorbed than for a solution of unlinked nucleotides. In single-stranded DNA, the bases are only partially shielded and will absorb an intermediate amount of light.
Highlighting sample contamination
Proteins have a maximum absorption of 280 nm, due to the amino acid tryptophan. Tryptophan possesses an aromatic ring, which makes this amino acid responsible for much of the light absorption by proteins. The relative purity of a sample can therefore be measured by testing the absorbance at both 260 and 280 nm and then calculating the ratio [2]. A value smaller than 1.8 indicates the presence of proteins and a value higher than 2.0 indicates probable contamination. Another common parameter used to describe the quality of DNA is the 260/230 nm ratio. This is used to estimate chemical contamination with compounds such as phenols and carbohydrates or a high salt concentration. The ideal 260/230 nm ratio is around 2.0.
By measuring at a wavelength at which the absorption level for nucleic acids and proteins is low, it is possible to correct for background readings caused by impurities. The wavelength most commonly used for this background subtraction is 320 nm; (Abs260-Abs320 / Abs280-Abs320). When bead-based purification is used, the 320 nm subtraction is always recommended.
Calculating DNA concentrations
The Lambert-Beer equation is used to measure how strongly a certain concentration of a substance absorbs light at a given wavelength i.e. the extinction coefficient. The average extinction coefficient for double-stranded DNA is 0.020 (μg/mL)-1 cm-1. This means that an absorption value of 1.0 at 260 nm corresponds to a concentration of 50 μg/mL. The amount of DNA in a sample can therefore be calculated by using the formula:
DNA concentration (μg/mL) = Abs260 x 50 μg/mL
Small sample sizes: overcoming the issue
The major challenge in calculating DNA concentrations is that sample sizes are often very low. The Thermo Scientific μDrop Plate enables measurements of samples at the microlitre (μL) scale. The μDrop Plate itself consists of two separate measurement locations: one area measures low sample volumes and the other area has been designed to hold traditional cuvettes [Figure 1].
The measurement area for low-volume samples consists of two quartz slides: the top slide is clear and the bottom one is partially Teflon-coated. The bottom slide contains 16 sample positions, arranged in a 2 x 8 matrix, on which samples can be easily pipetted.
Compared to a normal cuvette, the path length of the μDrop Plate low-volume area is really short; 10 mm vs. 0.5 mm respectively. By decreasing the path length, the sample volume can also be decreased. As a result, a sample volume down to 2 μL may be used with the μDrop Plate. DNA concentrations from a few nanograms to thousands of nanograms per microlitre can therefore be measured when a μDrop Plate is combined with a photometer displaying high precision and a wide linear range. Since the optical light path of the plate is 0.5 mm, a multiplication factor of 20 must be taken into account in the concentration calculations. The DNA concentration (μg/mL) can then be calculated by the following equation:
Abs260 x 50 μg/ml x (10 mm/0.5 mm) = Abs260 x 50 x 20
Putting the µDrop Plate to the test
An experiment was carried out to test the efficacy of the μDrop Plate at quantitating DNA concentrations and it’s consistency in comparison to cuvette based methods.
Methods
A DNA stock solution of Herring sperm DNA was serially diluted in a ratio of 1:2 into TE buffer (10 mM TRIS-HCl, 1 mM EDTA, pH 7.5). Seven different concentrations were created for testing. The concentrations of the samples were measured with the μDrop Plate low-volume area and the μDrop Plate cuvette position using:
• a Thermo Scientific Multiskan GO spectrophotometer
• a Thermo Scientific Varioskan Flash multimode reader
Concentrations were also measured with the Multiskan GO cuvette port and used as references for all the calculations.
The sensitivity of the assay was determined using two different parameters, Limit of Detection (LOD) and Limit of Quantification (LOQ) [3]. LOD is the lowest amount of analyte that can be separated from the background with statistical significance, but cannot necessarily be quantified as an exact value. LOQ is the lowest amount of the analyte at which quantification is possible with statistical relevance.
Results
1. Sensitivity
The results obtained with the μDrop Plate low volume area and the μDrop Plate cuvette area were compared to the results of the Multiskan GO cuvette port results [see reference 4 for more details]. Table 1 illustrates the LOD and LOQ levels of DNA that were measured by using the μDrop Plate low volume area.
2. Detection range
The μDrop Plate low-volume area measurements (measured with both a Multiskan GO and Varioskan Flash spectrophotometer) were compared with the reference readings of the DNA concentrations from the Multiskan GO cuvette port [Figure 2 and Figure 3].
A perfect correlation was also found when the cuvette measurements of the μDrop Plate were plotted against the Multiskan spectrophotometer cuvette port readings [Figure 4].
Stretching the capabilities of UV photometry
The results obtained demonstrate how low sample volumes can be used to accurately and sensitively quantitate the concentrations of DNA samples, from volumes as low as 2 µL with the Thermo Scientific μDrop Plate. The measurements with the μDrop low-volume area, taken using the Multiskan GO and the Varioskan Flash correlated well with the reference values, highlighting the systems’ reliability. In addition, the fixed path length of the plate enables a direct calculation of the nucleic acid concentration, allowing for greater flexibility to quantitate smaller volumes of precious samples.
References
1. Simoni RD, Hill RL, Vaughan M and Tabor H. A Classic Instrument: The Beckman DU Spectrophotometer and Its Inventor, Arnold O. Beckman. The Journal of Biological Chemistry 2003; 278: e1.
2. Stoscheck CM. Quantitation of Protein. Methods in Enzymology 1990; 182: 50-69.
3. Mocak J, Bond AM, Mitchell S and Scollary G. A statistical overview of standard (IUPAC and ACS) and new procedures for determining the limits of detection and quantification: Application to voltammetric and stripping techniques (Technical Report) 1997; 69 (2); 297-328.
4. Thermo Fisher Scientific (2011). Technical note: DNA quantification in micro-liter volumes with Thermo Scientific μDrop Plate. SP&A Application Laboratory, Thermo Fisher Scientific, Vantaa, Finland.
The author
Marika Raitio
Application Scientist
Thermo Fisher Scientific
