All of the following are correct regarding Southern blotting EXCEPT:

Explanation

## Southern Blotting: Principle and Limitations **Key Point:** Southern blotting has **limited sensitivity for detecting SNPs** because it relies on restriction fragment length polymorphism (RFLP) — a SNP must fall within a restriction site to alter fragment size. Most SNPs do not disrupt restriction sites and therefore cannot be detected by Southern blotting alone. ### Southern Blotting Workflow ```mermaid flowchart TD A[Genomic DNA]:::outcome --> B[Digest with restriction enzyme]:::action B --> C[Agarose gel electrophoresis]:::action C --> D[Separate by size]:::outcome D --> E[Transfer to membrane<br/>capillary/alkaline buffer]:::action E --> F[Hybridize with labeled probe]:::action F --> G[Detect hybridized bands]:::outcome ``` ### Correct Principles | Step | Method | Purpose | |------|--------|----------| | 1. Digestion | Restriction enzymes (EcoRI, BamHI, etc.) | Generate defined fragments | | 2. Separation | Agarose gel electrophoresis | Size-based separation | | 3. Transfer | Capillary blotting (alkaline buffer) | DNA → nitrocellulose/nylon membrane | | 4. Hybridization | Labeled probe (radioactive or fluorescent) | Detect complementary sequences | | 5. Detection | Autoradiography or fluorescence imaging | Visualize hybridized bands | **High-Yield:** Southern blotting detects: - **RFLPs** (restriction fragment length polymorphisms) — deletions, insertions, point mutations that alter restriction sites - **Copy number variations** — multiple bands indicate gene duplications; absent bands indicate deletions - **Gene rearrangements** — translocations, inversions **Warning:** Southern blotting does **NOT** reliably detect SNPs unless the SNP disrupts a restriction enzyme recognition site. Most SNPs are "silent" to restriction enzymes. Modern SNP detection requires **SNP arrays** or **sequencing**, not Southern blotting. **Clinical Pearl:** Southern blotting was historically used for diagnosing hemoglobinopathies (e.g., sickle cell disease, β-thalassemia) because the mutations alter restriction sites. However, it has been largely replaced by PCR-based methods and DNA sequencing in clinical practice.