Restriction Enzymes and RFLP

Sequence into size

A restriction enzyme cuts only at its recognition site (see the Foundations lesson on nucleases). A single-base change inside a site destroys it; a change that completes a site creates one. Either way the fragment sizes change, and a gel can see sizes ¹.

The PCR-RFLP workflow

1. Amplify a short region around the variant (100-500 bp).
2. Digest an aliquot with the chosen enzyme, in its buffer, at its temperature, for a set time.
3. Separate on agarose or polyacrylamide next to a ladder and an undigested sample.
4. Read the bands against the expected sizes worked out from the reference sequence ¹.

Reading the lanes

Example: a 300 bp amplicon with one site 100 bp from an end, destroyed by the variant.

If a variant creates a site, the pattern flips: normal stays uncut and the variant cuts. A heterozygote always shows every band from both homozygous patterns ¹. Real examples of each kind:

• Site destroyed: the sickle cell change in HBB abolishes an MstII site (historical Southern-blot test; later PCR-RFLP with DdeI).
• Site destroyed: Factor V Leiden, F5 c.1601G>A, abolishes an MnlI site in the common PCR-RFLP assay.
• Site created: HFE C282Y (c.845G>A) creates an RsaI site.

Controls and pitfalls

• Undigested lane: shows the full amplicon size.
• Known genotype controls: at least one that must cut completely. Incomplete digestion leaves some uncut product in a normal sample and looks just like a heterozygote - the most common RFLP error.
• Ladder: sizes every band.
• Small fragments: below about 50 bp they can run off or stain faintly; polyacrylamide resolves small differences better than agarose.
• Unexpected bands: a second, unrelated variant in the site (or a polymorphism creating a new one) can give a pattern you didn’t plan for.

Southern-blot RFLP

Before PCR, RFLP was done on genomic DNA with Southern blotting: digest micrograms of DNA, run it, transfer it to a membrane, and hybridize a labeled probe for the locus so only its fragments show ². It took days, needed micrograms of high-quality DNA, and often used radioactive probes ³. PCR-RFLP does the same job from nanograms in a day ¹. Southern blots survive for things PCR handles poorly, such as large FMR1 repeat expansions and their methylation status in fragile X.

Strengths and limits

Most labs now genotype single variants with real-time PCR probes or melt curves, and keep PCR-RFLP for confirmation, teaching, and low-volume tests.

What to take from this

A variant that destroys or creates a restriction site changes fragment sizes: amplify, digest, and run next to a ladder and uncut product. Normal and variant homozygotes give their own patterns and the heterozygote shows both (sickle cell and Factor V Leiden destroy sites; HFE C282Y creates one). Always include a control that must cut completely, because incomplete digestion mimics a heterozygote.