Amplified fragment length polymorphism (AFLP) is a fingerprinting technique that allows one to distinguish closely related species or subspecies that may be difficult or impossible to differentiate on the basis of morphological or biochemical characteristics. Like RAPD it requires no sequence information from the organism to be investigated, but obtaining the most useful data from AFLP analysis does involve some amount of optimization. Because stringent conditions for PCR are employed in AFLP, it is inherently more reproducible than the RAPD assay.

In essence, an AFLP fingerprint represents a subset of the population of restriction fragments resulting from a complete digest of the target genome. Characteristic differences in the fingerprints of two genomes result from polymorphisms that occur within or immediately adjacent to restriction sites, as well as from insertions and deletions occurring between restriction sites. The amount of information that would result if the entire population of restriction fragments were displayed would be overwhelming, and the AFLP procedure provides several means for adjusting the complexity of the information that is ultimately displayed on a gel.

The first step in AFLP is complete digestion of genomic DNA with restriction enzymes. In the analysis of plant genomes, where AFLP is currently finding wide use, the genomic DNA is digested with the "six-cutter" EcoRI and the "four-cutter" MseI. In a genome of 2 x 10⁹ bp, such a digestion would produce approximately 8 x 10⁶ fragments. MseI and EcoRI linkers are then ligated to the ends of the resulting fragments. These linkers provide the sequences for hybridization of primers in subsequent "pre-amplification" and AFLP-PCR steps. The "pre-amplification" step, in which PCR is performed using only a primer complementary to the EcoRI end, has been found useful for improving specificity. In the final AFLP-PCR step, primers complementary to EcoRI ends and MseI ends are used. Only the EcoRIprimer is labeled, however, so only EcoRI–EcoRI and EcoRI–MseI fragments are represented in the final AFLP pattern. The complexity of the pattern is reduced further through the use of primers containing one or more additional bases at the 3'-end. A single 3'-extension on both primers will reduce the number of fragments amplified by a factor of 16, a two-base extension will reduce the number by 256, and a three-base extension will reduce it by 4,096.

The challenge in optimizing AFLP is in obtaining an amount of information on the gel that is sufficient for the purpose, but not so great as to overcomplicate the analysis. This challenge is obviously related to the complexity of the target genome and the amount of genetic variability that exists within the group of organisms being examined. There are a number of steps at which the number of amplified fragments appearing on the gel can be limited:

	In the choice of restriction enzymes
	By including 3'-extensions on one or both linkers
	By including 3'-extensions on the "pre-amplification" primer
	In the labeling of the AFLP-PCR primers
	By including 3'-extensions on the AFLP-PCR primers

Unless one can apply an established procedure to the particular genome being investigated, a certain degree of "tuning" of the AFLP procedure must be expected.

AFLP analysis is usually performed on denaturing gels, and the fragments are visualized by either autoradiography or fluorescent imaging. For highly complex patterns, the length of a standard sequencing gel is required, but the resolution obtained with CleanGel is adequate for many purposes. Using silver staining for detection is in many cases advantageous over the use of radiolabeled primers.