Amplified fragment-length polymorphism analysis.

Norman Fry, Paul H.M. Savelkoul, Paolo Visca

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)89-104
Number of pages16
JournalMethods in molecular biology (Clifton, N.J.)
Volume551
DOIs
Publication statusPublished - 2009

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