Question
Explain the steps involved in DNA fingerprinting. What role do VNTRs play in making each person’s profile unique?
Solution — Step by Step
DNA is extracted from the biological sample — blood, hair follicle, saliva, semen, or tissue.
The extracted DNA is then cut at specific points using restriction enzymes (also called restriction endonucleases). These enzymes cut DNA at specific recognition sequences (palindromic sequences of 4-8 base pairs), producing DNA fragments of many different sizes.
VNTRs (Variable Number Tandem Repeats) are regions where a short DNA sequence is repeated head-to-tail, and the NUMBER of repeats varies from person to person. Because restriction sites flank the VNTR regions, cutting with restriction enzymes produces VNTR-containing fragments of different sizes in different individuals.
The DNA fragments are loaded into an agarose gel and an electric current is applied (gel electrophoresis).
DNA is negatively charged, so it moves toward the positive electrode. Smaller fragments move faster and travel farther. Larger fragments move slower.
After the run, the gel contains hundreds of DNA bands distributed by size. But they’re invisible at this stage.
The separated DNA in the gel is transferred to a nylon or nitrocellulose membrane by a technique called Southern blotting (invented by Edwin Southern in 1975).
The DNA is first denatured (made single-stranded) using an alkaline solution. Then the single-stranded DNA is transferred from the gel to the membrane by capillary action, maintaining the exact same pattern of bands.
The membrane is exposed to radioactively labelled DNA probes — short single-stranded DNA sequences complementary to the VNTR sequences.
The probes bind (hybridize) only to VNTR-containing fragments on the membrane. Unhybridized probe is washed away.
Only the bands corresponding to VNTR regions are now “tagged” with radioactivity.
The membrane is placed against X-ray film (autoradiography). The radioactive probes expose the film wherever they are located.
After developing the film, dark bands appear corresponding to the VNTR fragments. This pattern of bands — the DNA fingerprint — is unique to each individual (except identical twins).
Why This Works
VNTRs are useful for DNA fingerprinting because of individual variation in repeat number. If a person has 15 repeats at one VNTR locus, their fragment from that locus is a specific size. Another person with 8 repeats at the same locus has a smaller fragment. When we compare patterns from many VNTR loci simultaneously, the probability of two unrelated people having identical patterns at all loci is vanishingly small (less than ).
Alternative Method — PCR-Based Fingerprinting
Modern forensic labs mostly use STR (Short Tandem Repeat) analysis with PCR rather than the classic Southern blot method. PCR amplifies specific STR loci from tiny amounts of DNA, and fluorescent labels replace radioactive probes. Automated capillary electrophoresis gives results in hours. The principle is identical — comparing repeat number variation across multiple loci.
Common Mistake
Students often confuse VNTR with RFLP (Restriction Fragment Length Polymorphism). RFLP refers to differences in restriction sites between individuals. VNTR refers to differences in the number of tandem repeats. In DNA fingerprinting, both contribute — restriction enzymes are used to cut the DNA (RFLP aspect), and the variation in band sizes comes from VNTR differences. They are related but not identical concepts.