How we determine DNA Concentration?
The correct answer and explanation is :
DNA concentration is commonly determined using spectrophotometry or fluorometry. The two primary methods for measuring DNA concentration are:
- UV Spectrophotometry (Absorbance at 260 nm)
This method is based on the absorbance of ultraviolet (UV) light by nucleic acids, specifically at 260 nm. DNA absorbs UV light at this wavelength due to the presence of purine and pyrimidine bases. By measuring the absorbance at 260 nm (A260), you can determine the DNA concentration using Beer’s Law, which relates absorbance to concentration. The formula to calculate the DNA concentration is: $$
\text{DNA Concentration} (\mu g/mL) = \frac{A260 \times \text{Dilution Factor} \times 50}{1}
$$ where:
- $A260$ is the absorbance at 260 nm,
- The dilution factor accounts for any dilution of the sample,
- The factor 50 is the molar extinction coefficient for double-stranded DNA (dsDNA) in a 1 cm path length cuvette. Advantages:
- Rapid and inexpensive method.
- Can be used to estimate both the concentration and purity of DNA.
- It is a quick measurement that doesn’t require reagents, only a spectrophotometer. Limitations:
- The presence of contaminants like proteins (which absorb at 280 nm) or RNA can interfere with accurate measurements.
- It doesn’t provide detailed information about the integrity of the DNA.
- Fluorometry (using DNA-specific fluorescent dyes)
Fluorometric methods involve using DNA-specific fluorescent dyes (such as PicoGreen, SYBR Green, or Hoechst dyes), which bind specifically to DNA and fluoresce when exposed to light at specific wavelengths. The fluorescence intensity is proportional to the amount of DNA present, and the concentration is determined by comparing the fluorescence intensity to a standard curve. Advantages:
- More accurate than spectrophotometry in the presence of contaminants.
- Highly sensitive, allowing detection of low DNA concentrations. Limitations:
- Requires specific reagents, which can add to the cost.
- Fluorometers are more expensive than spectrophotometers.
Both methods have their uses depending on the DNA sample’s purity, the required accuracy, and available resources.