AFLP-PCR or simply AFLP is a PCR – based tool used in genetics research, DNA fingerprinting , and the practice of genetic engineering . Developed in the 1990s by Keygene, Amplified Fragment Length Polymorphism uses restriction enzymes to digest restriction fragments of genomic DNA , followed by ligation of the sticky ends to the adapters . A subset of the restriction fragments is then selected to be amplified. This selection is achieved by using primersComplementary to the adapter sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments (as described in detail below). Amplified fragments are separated and visualized upon denaturation on agarose gel electrophoresis , either via autoradiography or fluorescence method, or via automated capillary sequencing devices.
Although AFLP should not be used as an abbreviation, it is commonly referred to as “Amplified Fragment Length Polymorphism”. However, the resulting data are not scored as a length polymorphism, but as a presence–absence polymorphism. [2]
AFLP-PCR is a highly sensitive method for the detection of polymorphisms in DNA . The technique was originally described by Vos and Zabeau in 1993. [3] [2] In detail, the process of this technique is divided into three stages:
- Digestion of total cellular DNA with one or more restriction enzymes and binding of restriction half site specific adapters to all restriction fragments.
- Selective amplification of some of these fragments with two PCR primers containing the corresponding adapter and restriction site specific sequences.
- Electrophoretic separation of amplicons on a gel matrix , followed by visualization of the band pattern.
Application
AFLP technology has the potential to detect different polymorphisms in different genomic regions simultaneously. It is also highly responsive and reproducible. As a result, AFLP is widely used for the identification of genetic variation in strains or species of plants, fungi, animals, and bacteria. The AFLP technique has been used in criminal and paternity tests, as well as to quantify minor differences within populations, and to generate maps for quantitative trait locus (QTL) analysis in linkage studies.
AFLP has several advantages compared to other marker technologies, including randomly amplified polymorphic DNA ( RAPD ), restriction fragment length polymorphism (RFLP), and microsatellites . AFLP not only has higher reproducibility, resolution, and sensitivity at the whole-genome level than other techniques, [4] but also has the ability to expand to between 50 and 100 fragments at a time. Furthermore, no prior sequence information is required for amplification (Meudt & Clarke 2007). [5] As a result, AFLP has become extremely beneficial in the study of taxa including bacteria, fungi and plants, where much is still unknown about the genomic makeup of various organisms.
AFLP technology is covered by Keygene NV’s patents and patent applications AFLP is a registered trademark of Keygene NV
