title: A study of comparability in amplified fragment length polymorphism profiling using a simple model system.
abstract: A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.
authors: National Measurement Institute (NMIA), Pymble, NSW, Australia.
authors: Lina
authors: L
authors: Partis
authors: Malcolm
authors: M
authors: Burns
authors: Koichi
authors: K
authors: Chiba
authors: Philippe
authors: P
authors: Corbisier
authors: David
authors: D
authors: Gancberg
authors: Marcia J
authors: MJ
authors: Holden
authors: Jing
authors: J
authors: Wang
authors: Qing Yan
authors: QY
authors: Liu
authors: Tomoya
authors: T
authors: Okunishi
authors: Inchul
authors: I
authors: Yang
authors: Maxim
authors: M
authors: Vonsky
authors: Kerry R
authors: KR
authors: Emslie
mesh_terms: D004247:DNA / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D008962:Models, Theoretical; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
chemical_list: D004247:DNA