RFLP has been almost entirely replaced by PCR-based testing. The following description of RFLP is included here primarily for historic reasons (more current formats see below).
RFLP DNA testing has four basic steps:
1. The DNA from crime-scene evidence or from a reference sample is cut with something called a restriction enzyme. The restriction enzyme recognizes a particular short sequence such as AATT that occurs many times in a given cell's DNA. One enzyme commonly used is called Hae III (pronounced: Hay Three) but the choice of enzyme varies. For RFLP to work, the analyst needs thousands of cells. If thousands of cells are present from a single individual, they will all be cut in same place along their DNA by the enzyme because each cells DNA is identical to every other cell of that person.
2. The cut DNA pieces are now sorted according to size by a device called a gel. The DNA is placed at one end of a slab of gelatin and it is drawn through the gel by an electric current. The gel acts like a sieve allowing small DNA fragments to move more rapidly than larger ones.
3. After the gel has separated the DNA pieces according to size, a blot or replica of the gel is made to trap the DNA in the positions that they end up in, with small DNA fragments near one end of the blot and large ones near the other end. The blot is now treated with a piece of DNA called a probe. The probe is simply a piece of DNA that binds to the DNA on the blot in the position were a similar sequence (the target sequence) is located.
4. The size or sizes of the target DNA fragments recognized by the probe are measured. Using the same probe and enzyme, the test lab will perform these same steps for many people. These sizes and how they distribute among large groups of people form a database. From the database a rough idea of how common a given DNA size measured by a given probe is found. The commonness of a given size of DNA fragment is called a population frequency.
The restriction enzyme cuts the DNA into thousands of fragments of nearly all possible sizes. The sample is then electrophoretically separated. The DNA at this point is invisible in the gel unless the DNA is stained with a dye. A replica of the gel's DNA is made on something called a blot (also called a Southern blot) or membrane. The blot is then probed (mixed with) a special preparation of DNA that recognizes a specific DNA sequence or locus. Often, the probe is a radioactively labeled DNA sequence (represented by * labeled object in the figure above). Excess probe is washed off the blot, then the blot is laid onto X-ray film. Development reveals bands indicating the sizes of the alleles for the locus within each sample. The film is now called an "autorad." The band sizes are measured by comparing them with a "ladder" of known DNA sizes that is run next to the sample. A match may be declared if two samples have RFLP band sizes that are all within 5% of one another in size.For RFLP analysis to be reliable, all complex steps of the analysis must be carefully controlled. Databases must be large meaning they include many people; they must be representative of the potential test subjects. Because of the complexities of populations, databases must be interpreted with extreme care. For example, DNA fragment sizes rare in one population may be very common in other populations. Further, sub-populations or populations within populations must be considered.