The estimation of nucleic acids, specifically DNA and RNA, can be performed using the diphenylamine method for DNA and the orcinol method for RNA. Below is a detailed step-by-step explanation of each procedure.
(a) Estimation of DNA by the Diphenylamine Method
Principle
The diphenylamine method is based on the reaction between deoxyribose (the sugar component of DNA) and diphenylamine under acidic conditions. This reaction produces a blue complex that can be measured spectrophotometrically at 595 nm. The intensity of the color formed is proportional to the concentration of DNA in the sample.
Materials Required
- Commercial sample of DNA (10 mg)
- Buffer saline (0.15 M NaCl, 0.15 M sodium citrate made up to 500 mL)
- Diphenylamine reagent: dissolve 10 g of pure diphenylamine in 1 liter of glacial acetic acid and add 25 mL of concentrated H2SO4 (this solution must be prepared fresh)
- Boiling water bath
- Spectrophotometer
Procedure
- Preparation of Sample:
- Dissolve 10 g of nucleic acid in 50 mL of buffer saline.
- Pipette out 2 mL from this solution into a clean test tube.
- Addition of Reagents:
- To the test tube containing the nucleic acid solution, add 4 mL of diphenylamine reagent.
- Heating:
- Heat the mixture in a boiling water bath for approximately 10 minutes.
- Cooling:
- After heating, allow the mixture to cool to room temperature.
- Measurement:
- Measure the absorbance at 595 nm using a spectrophotometer against a blank prepared with buffer saline.
- Calculation:
- Use a standard curve generated from known concentrations of DNA to determine the concentration present in your sample based on its absorbance reading.
(b) Estimation of RNA by the Orcinol Method
Principle
The orcinol method relies on the reaction between pentose sugars (found in RNA) and orcinol in an acidic environment, which leads to the formation of furfural derivatives that react with orcinol to produce a colored complex measurable at 665 nm. This color change indicates the presence and concentration of RNA.
Materials Required
- RNA commercial sample (10 mg)
- Buffered saline
- Orcinol reagent: dissolve 10 g of ferric chloride (FeCl3·6H2O) in 1 liter of concentrated HCl and add 35 mL of 6% w/v orcinol in alcohol.
- Boiling water bath
- Spectrophotometer
Procedure
- Preparation of Standard Solutions:
- Prepare standard RNA solutions ranging from 0–2 mL by diluting commercially available RNA (0.2 mg/mL) with distilled water up to a total volume of 2 mL.
- Addition of Orcinol Reagent:
- To each test tube containing standard solutions, add 3 mL of orcinol reagent.
- Heating:
- Heat all tubes in a boiling water bath for about 20 minutes.
- Cooling:
- Allow the tubes to cool down to room temperature after heating.
- Measurement:
- Measure the optical density at 665 nm against an orcinol blank using a spectrophotometer.
- Calculation:
- Determine the concentration of RNA present in your samples based on their absorbance readings compared to those from standard solutions.
In summary, both methods provide reliable means for estimating nucleic acids through colorimetric reactions that can be quantified using spectrophotometry, allowing researchers to assess nucleic acid concentrations effectively in various biological samples.
