RFLP has been 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 basic steps:
The DNA from crime-scene facts or from a reference sample is cut with something called a restriction enzyme. The restriction enzyme recognizes a specific short sequence such as AATT that occurs lots of times in a given cell's DNA. enzyme often used is called Hae III (pronounced: Hay) 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.
The cut DNA pieces are now sorted according to size by a gizmo called a gel. The DNA is placed at finish of a slab of gelatin & it is drawn through the gel by an electric current. The gel acts like a sieve allowing tiny DNA fragments to move more quickly than larger ones.
The size or sizes of the target DNA fragments recognized by the probe are measured. Using the same probe & enzyme, the check lab will perform these same steps for lots of people. These sizes & how they distribute among massive 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.
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 finish up in, with tiny DNA fragments near finish of the blot & massive ones near the other finish. The blot is now treated with a piece of DNA called a probe. The probe is basically a piece of DNA that binds to the DNA on the blot in the position were a similar sequence (the target sequence) is located.
For RFLP analysis to be reliable, all complex steps of the analysis must be carefully controlled. Databases must be massive meaning they include lots of people; they must be representative of the potential check subjects. Because of the complexities of populations, databases must be interpreted with extreme care. For example, DNA fragment sizes rare in population may be common in other populations. Further, sub-populations or populations within populations must be thought about.
The restriction enzyme cuts the DNA in to thousands of fragments of all feasible 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 labelled DNA sequence (represented by * labelled 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 samples have RFLP band sizes that are all within 5% of another in size.
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