Nucleic Acid Quantification
Related: DNA Extraction · RNA Extraction · NGS Library Preparation · qPCR
1. Principle
Quantification and purity assessment estimate how much DNA/RNA is present and whether contaminants will impair PCR or sequencing — using UV absorbance, fluorescence dyes, or electrophoretic sizing.
2. Step-by-Step Procedure (conceptual)
- Choose method by purpose: UV screen vs fluorometric accuracy vs fragment sizing.
- Calibrate / use kit standards (fluorometry).
- Measure concentration and purity ratios; for NGS, also check fragment length.
- Normalize input mass/moles into PCR or NGS Library Preparation.
3. Interpretation
| Method | Measures | Best for | Limits |
|---|---|---|---|
| NanoDrop / UV | A260, A260/280, A260/230 | Quick purity screen | Overestimates; reads free NTPs, ssDNA, RNA |
| Qubit / PicoGreen | Dye-specific dsDNA/RNA | NGS input | No fragment size |
| Bioanalyzer / TapeStation | Size + approx. conc. | Libraries, RNA integrity | Cost |
| qPCR / Digital PCR | Amplifiable copies | Functional titer | Assay-specific |
4. Clinical Use Cases
- Gatekeeping extracts before expensive sequencing.
- Normalizing multiplex panel inputs.
- Troubleshooting weak/failed PCR (low input vs inhibition).
5. Comparison with Other Methods
UV is fastest; fluorescence is most trustworthy for dsDNA mass; dPCR counts molecules when absolute quantification matters.
6. Mnemonic / Visual Aid
UV sees everything · Dye sees what you care about · Electrophoresis sees the shape.
Active Recall
- Ideal A260/280 for pure DNA?
- Why can NanoDrop say “enough DNA” while library prep fails?
- When prefer dPCR over Qubit?