Question

Explain the technique of DNA fingerprinting by Southern-blot.

Answer 1

DNA fingerprinting by Southern blotting detects polymorphic DNA regions, commonly VNTRs, and reveals an individual-specific pattern of bands.

Procedure

1. Isolation: Extract and purify DNA from a nucleated-cell sample. If the sample is very small, DNA may first be amplified by PCR.
2. Restriction digestion: Cut the DNA into fragments using restriction endonucleases.
3. Electrophoresis: Separate the fragments by size through a gel. Smaller fragments move farther than larger ones.
4. Denaturation and blotting: Make the DNA fragments single-stranded, then transfer them from the gel onto a nylon or nitrocellulose membrane by Southern blotting.
5. Hybridisation: Add labelled single-stranded DNA probes complementary to the target VNTR sequences. The probes bind to matching fragments.
6. Detection: Expose the membrane to an X-ray film (autoradiography). The detected bands form a pattern that can be compared between samples.

Because VNTR patterns vary greatly among individuals, the resulting banding pattern can be used for identification and biological relationship testing.