DNA Extraction
Related MOCs: MOC - Diagnostic & Lab Methods
Related: RNA Extraction · Nucleic Acid Quantification · PCR · Whole-Genome Sequencing
1. Principle
DNA extraction isolates and purifies genomic (and sometimes plasmid/extracellular) DNA from cells or specimens by lysing organisms, removing proteins/lipids/inhibitors, and recovering DNA suitable for amplification or sequencing.
2. Step-by-Step Procedure (conceptual)
- Lysis — mechanical (bead-beating), enzymatic (lysozyme, proteinase K), chemical (detergents, chaotropes), or heat; hardeners for spores/mycobacteria/fungi.
- Inactivation of nucleases — chaotropic salts, heat, proteinase K.
- Separation — spin columns (silica), magnetic beads, organic extraction (phenol-chloroform, less common now), or automated cartridges.
- Wash — remove salts, proteins, PCR inhibitors (heme, humic acids, bile).
- Elute — low-salt buffer or water; quantify/qualify (Nucleic Acid Quantification).
- Proceed to PCR, qPCR, library prep, or storage (−20/−80 °C).
3. Interpretation / QC
- Adequate yield & purity: A260/280 ≈ 1.8; A260/230 free of salt/organics; fluorometric dsDNA preferred for NGS.
- Pitfalls: Incomplete lysis (false-negative PCR); inhibitors left behind; cross-contamination between high-load samples; plasmid DNA may need separate protocols.
4. Clinical Use Cases
- Upstream of nearly all NAATs and WGS from isolates and primary specimens.
- Hard-to-lyse targets: Mycobacterium, Gram-positives, fungi, spores — protocol choice matters.
- Blood culture bottles, swabs, tissue, stool — each has preferred kits/workflows.
5. Comparison with Other Methods
| Approach | Pros | Cons |
|---|---|---|
| Silica spin column | Clean DNA, familiar | Manual steps, clogging |
| Magnetic beads | Automatable, scalable | Beads/carryover if poorly washed |
| Crude boil prep | Fast, cheap | Inhibitors; not for NGS |
| RNA Extraction | Needed for RNA viruses | RNase risk; different chemistry |
6. Mnemonic / Visual Aid
Break → Bind → Wash → Elute — the four beats of modern DNA prep.
Related Concepts
- Sample Types and Specimen Quality · NGS Library Preparation · Metagenomic NGS
- Bioinfo next: Read QC and Preprocessing · WGS Bioinformatics Pipeline
Active Recall
- Why might a Gram-positive fail PCR after a “soft” lysis kit?
- Why prefer fluorometry over NanoDrop for NGS input?
- Name two common PCR inhibitors in clinical specimens.