Amplified Fragment Length Polymorphism Analysis

Amplified fragment length polymorphism (AFLP) analysis is a universal polymerase chain reaction (PCR)-based DNA fingerprinting technique comprising three main stages: (i ) digestion of genomic DNA with restriction endonucleases and ligation to double-stranded adaptors (each comprised of two oligonucleotides), thus creating restriction fragments with identical known adaptor sequences; (ii ) specific amplification of a subset of these DNA fragments using primers (one labeled) targeting the adaptor sequences and additional selected bases within the unknown genomic DNA; and (iii ) analysis of the patterns (usually automated). Differences or polymorphisms between samples are revealed by separation of the labeled fragments by electrophoresis (standard agarose, high-resolution denaturing acrylamide, or capillary gels). Comparison of banding patterns is typically achieved using dedicated fingerprinting analysis software. The advantages of AFLP analysis include the ability to use a universal protocol in combination with different restriction endonucleases and the choice of adding one or more selective nucleotides in the PCR �primers to achieve optimal results relatively quickly without prior knowledge of DNA sequences from a large variety of (micro)organisms. The method also has the potential for high-throughput and local electronic database pattern storage with relatively low cost. Disadvantages include variation in the precision of sizing of fragments, leading to suboptimal reproducibility, particularly across different platforms.