Restriction Enzymes and RFLP

Overview

The foundations lesson on nucleases and DNA ligase introduced restriction endonucleases as reagents that recognize a short, defined sequence and cut the DNA backbone wherever that sequence occurs. This lesson applies that property to a detection problem: using restriction digestion to tell two versions of a sequence apart. The technique is restriction fragment length polymorphism (RFLP), one of the earliest ways to read a specific sequence difference without sequencing the DNA directly.

The core idea is small but powerful. A restriction enzyme converts sequence information into fragment-size information. If a sequence variant changes whether a recognition site is present, it changes the sizes of the fragments produced — and fragment sizes are easy to measure. RFLP is the method that turns that fact into a genotype.

A brief recap of restriction enzymes

Only the points needed here are repeated; the full treatment is in the foundations lesson. A restriction endonuclease binds a specific recognition site, usually a palindrome of four to eight base pairs, and cleaves both strands at or near that site ¹. Depending on where the strands are cut relative to the center, the enzyme leaves blunt ends (a flush cut) or sticky ends (a staggered cut with short single-stranded overhangs). For RFLP the kind of end does not matter; what matters is whether the site is present at all, because that determines whether the enzyme cuts.

How a sequence variant changes fragment sizes

A recognition site is just a specific run of bases. A single-base change can therefore have an all-or-nothing effect on the enzyme:

• A variant that destroys a site removes a cut the enzyme would otherwise make. The two fragments that would have been produced stay joined as one larger fragment.
• A variant that creates a site adds a cut where there was none, splitting one fragment into two smaller ones.

In both cases the total length of DNA is unchanged; only the pattern of cuts differs. Because electrophoresis separates fragments by size, that difference in cut pattern becomes a visible difference in band positions. A sequence change that alters a restriction site in this way is the polymorphism in “restriction fragment length polymorphism” ¹.

The method only works when the variant of interest happens to lie in — or happens to create — a restriction site for some available enzyme. That requirement is the central limitation of the technique, returned to below.

The PCR-RFLP workflow

In modern practice RFLP is almost always performed on a PCR product rather than on whole genomic DNA, which keeps the analysis focused on one small region. The workflow has four steps:

1. Amplify. Use PCR to copy the region containing the candidate variant, producing many identical copies of a defined length. (PCR is developed in the amplification module; here it simply provides abundant, uniform template.)
2. Digest. Add a restriction enzyme chosen because its recognition site coincides with the variant — that is, the site is present for one allele and absent for the other ¹.
3. Separate. Run the digest on a gel so the resulting fragments line up by size. (Electrophoresis is the subject of the next module; for now treat it as a size ruler that sorts fragments largest-to-smallest from the well.)
4. Interpret. Read the band pattern and infer the genotype: an uncut product indicates the allele lacking the site, while cut products indicate the allele carrying the site.

The choice of enzyme in step 2 is the crux of assay design. The analyst inspects the sequence around the variant and selects an enzyme whose recognition site is made or broken by the base change ².

A worked conceptual example

Consider a 300 bp PCR product. Suppose the wild-type allele contains a single restriction site located 100 bp from one end. Digestion therefore cuts the 300 bp product into two fragments: 100 bp and 200 bp.

Now suppose a variant allele carries a single-base change that falls inside that recognition site and abolishes it. The enzyme no longer cuts, so the variant product stays intact at its full 300 bp.

                       cut here (100 bp from left end)
                              |
wild-type (site present):  [===100===|=========200=========]
                           digest -> 100 bp + 200 bp (two fragments)

variant (site abolished):  [================300================]
                           digest -> 300 bp (uncut, one fragment)

The two alleles are now distinguishable purely by fragment size. On a gel the fragments migrate to size-dependent positions (larger fragments stay near the well, smaller fragments travel farther):

   well (top)
   ===========
   |  300  |   <- variant band (uncut)
   |  200  |   <- wild-type band
   |  100  |   <- wild-type band
   v (smaller fragments migrate farther)

This single-site example is deliberately generic; the same logic scales to real assays, where the analyst confirms the expected fragment sizes against the known sequence before interpreting any sample.

Reading homozygous and heterozygous patterns

Because a person carries two copies of most autosomal loci, the digest reflects both alleles at once. Three patterns are possible for the example above:

• Homozygous wild-type (site present on both copies): only the cut fragments appear — bands at 200 bp and 100 bp.
• Homozygous variant (site absent on both copies): only the uncut product appears — a single band at 300 bp.
• Heterozygous (one allele of each): all three bands appear — 300, 200, and 100 bp — because one copy is cut and the other is not.

              homozygous WT   homozygous variant   heterozygous
   300 bp          -                 ===                ===
   200 bp         ===                 -                 ===
   100 bp         ===                 -                 ===

The heterozygote shows the combined pattern of both homozygotes — a useful check when interpreting results, since a true heterozygous lane should contain every band the two homozygous patterns contribute ¹. A control lane of undigested product and a size marker (ladder) are run alongside so that “no cut” can be distinguished from “digestion failed.”

The historical Southern-blot RFLP

Before PCR was widespread, RFLP was performed directly on genomic DNA and read by Southern blotting ³. At a high level: genomic DNA was digested with a restriction enzyme, the large mixture of fragments was separated by electrophoresis, transferred from the gel onto a membrane, and then probed with a labeled fragment specific to the locus of interest. The probe revealed only the bands containing its target sequence, so a site-altering variant showed up as a shift in those bands. This approach required large amounts of DNA, took days, and used labeled probes; PCR-RFLP later achieved the same genotyping from a tiny sample in a single day, which is why the PCR-based form dominates today ¹. Probe hybridization and blotting are developed with the detection techniques later in the program.

Strengths and limitations

RFLP earns continued use for a few reasons:

• It is simple and inexpensive, requiring only PCR, a single enzyme, and a gel — no specialized instrument.
• The readout is direct and easy to interpret for a single known variant, and it doubles as a clear illustration of how sequence maps to fragment size.

Its limitations are equally clear:

• It is low-throughput: one or a few variants per reaction, read by eye.
• It works only when a restriction site coincides with the variant. Many variants do not create or destroy any convenient site, leaving no enzyme able to distinguish the alleles ².
• It reports only the specific change the enzyme tests for; it cannot reveal other nearby variants, and an unexpected mutation inside the recognition site can cause an enzyme to fail to cut and be misread.

When these constraints bind, other approaches are preferred. Sequencing reads the bases directly and is not restricted to convenient sites, and real-time (probe-based) methods genotype many samples quickly without a gel; both are covered later in the program by topic. RFLP remains valuable as a low-cost confirmatory test and as a clear demonstration that a restriction enzyme converts sequence into size.