Amplified fragment length polymorphism

Amplified fragment length polymorphism PCR, or "AFLP-PCR" (often AFLP), is a tool used in the study of genetics and in the practice of genetic engineering.

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Amplified Fragment Length Polymorphism (AFLP) is a polymerase chain reaction (PCR) based genetic fingerprinting technique that was developed in the early 1990’s by Keygene. AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments. A subset of the restriction fragments are then amplified using 2 primers complementary to the adaptor and restriction site fragments. The fragments are visualized on denaturing polyacrylamide gels either through autoradiographic or fluorescence methodologies.

AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA.The technique was originally described by Vos and Zabeau in 1993. The procedure of this technique is divided into three steps:

1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

A variation on AFLP is TE Display, used to detect transposable element mobility.

Applications

  The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As a result, AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. The AFLP technology has been used in criminal and paternity tests, in population genetics to determine slight differences within populations, and in linkage studies to generate maps for QTL analysis.

There are many advantages to AFLP when 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, but it also has the capability to amplify between 50 and 100 fragments at one time. In addition, no prior sequence information is needed for amplification. 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.

See also

• Restriction fragment length polymorphism (RFLP)

References

• Zabeau, M and P. Vos. 1993. Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Office, publication 0 534 858 A1, bulletin 93/13.
• Vos, P., Hogers, R., Bleeker, M., et al. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23(21):4407-4414 [1]
• Van den Broeck et al., 1998
• Casa et al., 2000
• Biedler et al., 2003
• Ulrich G. Mueller and LaReesa Wolfenbarger. 1999. AFLP Genotyping and fingerprinting.Tree. Vol14. 10:389 - 394.
• O’Shea, B., Khare, S., Bliss, K., Klein, P., Ficht, T. A., Adams, L. G., Rice-Ficht, A. C. Genotyping and Characterization of Mycobacterium avium subsp. paratuberculosis Using Amplified Fragment Length Polymorphism. 2004. Journal of Clinical Microbiology. 42:3600-3606