Southern blot

**Steps (SNoW DRoP mnemonic: Southern = DNA, Northern = RNA, Western = Protein):** 1. Isolate DNA from the sample (blood, tissue). 2. Digest with restriction endonucleases to produce fragments. 3. Separate fragments by size using agarose gel electrophoresis (smaller fragments migrate farther toward the anode/positive pole because DNA is negatively charged). 4. Denature the DNA (alkaline treatment) to make it single-stranded. 5. Transfer (blot) to a nitrocellulose or nylon membrane by capillary action. 6. Hybridize with a labeled (radioactive or fluorescent) single-stranded DNA probe complementary to the target sequence. 7. Wash off unbound probe and visualize (autoradiography or fluorescence). Bands appear only where the probe binds the complementary sequence.

**Clinical uses:** - Detecting large deletions, insertions, and gene rearrangements (e.g., B- and T-cell receptor/immunoglobulin gene rearrangements for clonality in lymphoid malignancies). - Diagnosing trinucleotide repeat expansion disorders, especially Fragile X syndrome (CGG repeats in FMR1). Methylation status can also be assessed with methylation-sensitive restriction enzymes. PCR is used first for smaller expansions, while Southern blot is useful for very large expansions. - RFLP (restriction fragment length polymorphism) analysis for DNA fingerprinting, paternity testing, and linkage analysis. - Detecting gene amplification and structural variants (e.g., historically for BCR-ABL rearrangement, and for sickle cell disease before PCR methods). - Confirming transgene integration in research.

**Comparison of blots:** - Southern: DNA sample, DNA probe. - Northern: RNA sample (gene expression), DNA or RNA probe. - Western: protein sample, labeled antibody probe (e.g., HIV confirmation, historically). - Southwestern: DNA-binding proteins (e.g., transcription factors), detected using labeled DNA probe.

**Key points for boards:** - Named after Edwin Southern (the others were named by wordplay on his name, so they are not named after people). - Requires prior knowledge of the target sequence for probe design. - Detects the presence, size, and copy number of a DNA fragment, not its expression. - Related techniques: PCR (amplifies DNA), FISH (localizes sequences on chromosomes), microarray, and next-generation sequencing, which have largely replaced Southern blot in many diagnostic settings.