AFLP®
Genetic Fingerprinting
The AFLP (Amplified Fragment Length Polymorphism) 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
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). template concentration, PCR cycle profile).
Figure 2. The selective principle of the AFLP technology.
Benefits
- achieving of high marker density: 50 – 100 amplified fragments per AFLP fingerprint
- detectable polymorphisms because of presence or size of restriction fragments
- widely used because of a wide scope of restriction enzymes
- no prior sequence information needed
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.