The AFLP® technology was developed by KeyGene in the early 1990’s and has become one of the most popular genetic fingerprinting technologies world-wide.
How it works
For an introductory AFLP video please click here:
AFLP is a DNA fingerprinting technique which detects multiple DNA restriction fragments by means of PCR amplification. The AFLP technology usually comprises of the following steps:
the restriction of the DNA with two restriction enzymes, preferably a hexa-cutter and a tetra-cutter;
the ligation of double-stranded adapters to the ends of the restriction fragments;
the amplification of a subset of the restriction fragments using two primers complementary to the adapter and restriction site sequences, and extended at their 3' ends by "selective" nucleotides (Figures 1 and 2);
gel electrophoresis of the amplified restriction fragments on denaturing slab gels or cappilaries;
the visualization of the DNA fingerprints by means of autoradiography, phospho-imaging, or other methods.
Figure 1. An overview of the AFLP technology
The amplification primers, known as AFLP primers, are generally 17 - 21 nucleotides in length and anneal perfectly to their target sequences; i.e. the adapter and restriction sites, and a small number of nucleotides adjacent to the restriction sites (Figure 2). This renders AFLP a very reliable and robust technique, which is unaffected by small variations in amplification parameters (e.g. thermal cyclers, template concentration, PCR cycle profile).
Figure 2. The selective principle of the AFLP technology.
A major advantage of the technology is the high marker density that can be obtained. A typical AFLP fingerprint contains between 50 and 100 amplified fragments. The frequency with which AFLP markers are detected depends on the level of sequence polymorphism between the tested DNA samples. The molecular basis of AFLP polymorphisms will usually be single nucleotide polymorphisms in the restriction sites, or in the selective nucleotides adjacent to the restriction sites (causing the AFLP primers to mispair at the 3' end). In addition, deletions, insertions and rearrangements affecting the presence or size of restriction fragments will result in detectable polymorphisms.
The AFLP technology can be applied using a wide scope of restriction enzymes and all feasible combinations of selective nucleotides. Depending on the degree of polymorphism between samples, individual samples can be fingerprinted using more and/or different enzyme and primer combinations.
The application of the AFLP technology requires no prior sequence information. If sequence information is available an in-silico analysis can be done to select the most informative enzyme and primer combinations. In-silico analyses can also support physical and genetic mapping projects.
APPLICATIONS
Genetic Diversity Analysis
Variety Identification
Acceleration of Inbred Conversions
Removal of Linkage Drag
Linked Marker assay Development
Seed and Plant Quality Assay Development
Introgression Line Library Construction
Genetic Map Construction
Figure 3. An example of an AFLP fingerprint
For a more detailed description of the technology:
Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; Lee, Th. van der; Hornes, M.; Frijters, A.; Pot, J.; Peleman, J.; Kuiper, M. & Zabeau, M. (1995).
AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Research 23(21): 4407-4414.
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