What Are Key Laboratory Techniques to Distinguish DNA from RNA?

In the lab, several techniques are used to distinguish between DNA and RNA by exploiting their fundamental differences in size, chemical stability, and susceptibility to enzymes.

1. Enzymatic Digestion (DNase/RNase Test)
   • Principle: Uses enzymes that are highly specific. DNase (Deoxyribonuclease) specifically degrades DNA. RNase (Ribonuclease) specifically degrades RNA.
   • Procedure: Take three samples of your nucleic acid. Add DNase to one, RNase to another, and nothing to the third (control). Then, run all three on a gel.
   • Result: If the nucleic acid band disappears in the DNase-treated lane, it was DNA. If it disappears in the RNase-treated lane, it was RNA.
2. Alkaline Hydrolysis
   • Principle: Exploits the chemical instability of RNA (its 2'-OH group).
   • Procedure: Expose the sample to a strong alkaline (basic) solution, such as sodium hydroxide (NaOH).
   • Result:RNA will rapidly degrade (hydrolyze) into small fragments. DNA will remain stable (its strands will separate, or "denature," but the backbone will not break).
3. Agarose Gel Electrophoresis
   • Principle: Separates molecules by size.
   • Procedure: Load the extracted nucleic acid sample into an agarose gel and apply an electric current.
   • Result: Intact genomic DNA (gDNA) is enormous and will appear as a single, very large band at the top of the gel. RNA will appear as two distinct, bright bands representing the large (28S) and small (18S) subunits of ribosomal RNA (rRNA), which are much smaller and travel further down the gel.
4. UV Spectrophotometry (A260/280 Ratio)
   • Principle: Measures the purity of a nucleic acid sample.
   • Procedure: Use a spectrophotometer to measure how much UV light the sample absorbs at 260 nm (the peak for nucleic acids) and 280 nm (the peak for proteins).
   • Result: A pure DNA sample has an A260/280 ratio of approximately 1.8. A pure RNA sample has an A260/280 ratio of approximately 2.0. This ratio can quickly indicate what is *likely* in the sample.