| Abstract |
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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.
Affiliation(s): (2) Respiratory and Systemic Infection Laboratory, Health Protection Agency, Centre for Infections, London, UK
(3) Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
(4) Department of Biology, University Roma Tre, and Molecular Microbiology Unit, National Institute for Infectious Diseases “Lazzaro Spallanzani”, Rome, Italy
(3) Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
(4) Department of Biology, University Roma Tre, and Molecular Microbiology Unit, National Institute for Infectious Diseases “Lazzaro Spallanzani”, Rome, Italy
Series: Methods in Molecular Biology | Volume: 551 | Pub. Date: Feb-01-2009 | Page Range: 89-104 | DOI: 10.1007/978-1-60327-999-4_8
Subject: Cell Biology
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