Amplified Fragment Length Polymorphism: Applications and Recent Developments
AFLP or amplified fragment length polymorphism is a PCR-based molecular technique that uses selective amplification of a subset of digested DNA fragments from any source to generate and compare unique fingerprints of genomes. It is more efficient in terms
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Introduction The AFLP technique is a patented technology first described by [1] and is applied widely in monitoring inheritance of agronomic traits in plants, pedigree analysis, parentage analysis, screening of DNA markers linked to genetic traits and genes of interest, etc. AFLP technique uses the entire genome for polymorphism and reproducibility and is recognized as a universal DNA fingerprinting system, universally accepted regarding origin and complexity of DNA samples and even small sequence variations that can be identified using a small quantity of DNA as low as 0.05 μg. A large number of
Pascale Besse (ed.), Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology, vol. 2222, https://doi.org/10.1007/978-1-0716-0997-2_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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fragments are detected on a gel that allows evaluation of a large number of loci at a time. It is much advantageous in terms of number of polymorphisms identified per reaction, reproducibility, ease, and cost of analysis. 1.1
Principle of AFLP
1.2 Basic Steps Involved in AFLP Analysis
AFLP employs selective amplification of restriction fragments from a digested total genomic DNA using PCR. Genomic DNA is first digested by two restriction enzymes that cut the big molecules into a mixture of fragments enabling amplification by PCR. Usually in AFLP two restriction enzymes, a rare cutter like EcoRI (6-bp restriction site) and a frequent cutter like MseI (4-bp restriction site), are used for restriction. Double-stranded oligonucleotide adapters consisting of a core sequence and a restriction enzymespecific sequence homologous to one 50 or 30 end are then ligated to the DNA fragments using T4 DNA ligase. The ligated DNA fragments are amplified by PCR using primers complementary to the adapter and restriction site sequence with additional selective nucleotides at their 30 end. Using selective primers reduces the complexity of the mixture, and those fragments complementary to nucleotides beyond restriction site will be amplified by these selective primers under stringent annealing conditions. Later, the polymorphisms are identified by a denaturing polyacrylamide gel electrophoresis and patterns between individuals are compared. In AFLP polymorphisms observed arise due to a mutation in the restriction site, a mutation in the regions complementary to primer extensions and adjacent to restriction site, or a deletion/insertion within the amplified region. Molecular polymorphisms are identified based on the presence or absence of particular DNA fragments of a given size among individuals (Fig. 1). A suitable DNA extraction protocol that yields good quality DNA without degradation may be employed for AFLP analysis. Quality of DNA needs to be ensured by an extra purification step; in case if the DNA extracts contain restriction or PCR inhibitors, an extra purification step may be incorporated. In AFLP, it is required to optimize the quantity of DNA for generating c
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