DNA concentration measures the amount of deoxyribonucleic acid in a sample. Accurate DNA quantification is important for various biological and scientific applications, ensuring consistent and reproducible results in experiments like polymerase chain reaction (PCR), cloning, and next-generation sequencing. Without this precise measurement, subsequent molecular biology workflows can be compromised.
Measuring DNA Using Light Absorption
Spectrophotometry is a common method for measuring DNA concentration, utilizing the principle that nucleic acids absorb ultraviolet (UV) light. A spectrophotometer passes light through a sample, measuring how much is absorbed, with DNA absorbing most strongly at 260 nanometers (A260) due to its nucleotides. The Beer-Lambert Law explains that the amount of light absorbed is directly proportional to the substance’s concentration, meaning a higher A260 reading indicates more DNA. For accurate readings, the A260 value should typically fall within the instrument’s linear range, generally between 0.1 and 1.0.
Translating Measurements into Concentration
A standard formula calculates DNA concentration from A260 readings; for double-stranded DNA (dsDNA), an A260 reading of 1.0 corresponds to 50 µg/mL. The formula to determine DNA concentration in micrograms per milliliter (µg/mL) is: Concentration (µg/mL) = (A260 reading) × (dilution factor) × (50 µg/mL). The “dilution factor” accounts for any dilution made to the original DNA sample before measurement, ensuring the reading falls within the linear range. For example, if a diluted DNA sample has an A260 reading of 0.5 and was diluted 10-fold, the concentration would be 0.5 × 10 × 50 µg/mL = 250 µg/mL. DNA concentration is often expressed in nanograms per microliter (ng/µL), where 1 µg/mL is equivalent to 1 ng/µL.
Assessing DNA Purity and Common Issues
While the A260 reading provides DNA concentration, other absorbance readings offer insights into sample purity. The A260/A280 ratio assesses protein contamination; pure DNA typically has a ratio between 1.7 and 2.0. A ratio lower than 1.7 may suggest protein or phenol contamination, as proteins absorb strongly at 280 nm.
The A260/A230 ratio indicates contamination by organic compounds (e.g., salts, carbohydrates, organic solvents). For pure DNA, this ratio is generally expected to be higher than the A260/A280 ratio, often in the range of 1.5 to 2.2. A low A260/A230 ratio signifies these contaminants, which can interfere with accurate A260 measurements and downstream applications. Readings at 320 nm can also indicate turbidity or particulate contamination.
Other Approaches to DNA Quantification
Beyond spectrophotometry, several alternative methods are available for DNA quantification. Fluorescence-based assays use specific dyes that bind to DNA and emit fluorescence, which is then measured by a fluorometer. These methods offer higher sensitivity and are particularly specific for double-stranded DNA, making them suitable for low-concentration samples. However, they typically require a standard curve generated from DNA samples of known concentrations for accurate quantification.
Another method is agarose gel electrophoresis, which separates DNA fragments by size and allows for visual estimation of concentration and assessment of DNA integrity. While not as precise for exact quantification as spectrophotometry or fluorometry, it helps confirm the presence of DNA and evaluate its quality visually. Quantitative Polymerase Chain Reaction (qPCR) is also used for DNA quantification, offering high specificity and sensitivity by amplifying and detecting specific DNA sequences.