Molecular Techniques and Methods

Amplified Fragment Length Polymorphisms (AFLPS)

Copy Right © 2001/ Institute of Molecular Development LLC

Genomic DNA is digested with a restriction enzyme and then double-stranded oligonudeotide adapters are ligated to the ends of the fragments. A subset of fragments is then amplified by PCR using primers designed to include part of the adapter, the restriction site and about three nudeotides beyond the site. Since only a proportion of the DNA fragments will display complementarity to these three 3'-nudeotides, only this subset of fragments will be amplified. A non-proofreading enzyme such as Taq DNA polymerase must be used to ensure that the specificity of the 3'-end of the primer is maintained. The result is that many fragments will be simultaneously amplified and a radiolabeled dNTP can be included to label the products which can be separated by electrophoresis through a DNA sequencing gel. The approach can be used to compare samples that display phenotypic traits, and bands that appear in one phenotype but not the other may represent markers linked to the phenotypic marker. The use of AFLP markers can provide powerful tools for molecular breeding strategies in both plants and animals. The AFLP approach has also been used for comparative analysis of cDNA populations, thus allowing differential analysis of expressed genes.




REFERENCES

  • Meksem, K, Leister, D, Peleman, J, Zabeau, M, Salamini, F, Gebhardt, C (1995) A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers. Mol. Gen. Genet. 249: 74-81.

  • Thomas, CM, Vos, P, Zabeau, M, Jones, DA, Norcott, A, Chadwick, BP, Jones, JDG (1995) Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9 gene for resistance to Cladosporium fulvum. The Plant J. 8(5): 785-794.

  • Vos, P, Hogers, R, Bleeker, M, Reijans, M, van de Lee, T, Hornes, M, Frijters, A, Pot, J, Peleman, J, Kuiper, M, Zabeau, M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23(21): 4407-4414.



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