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Amplified fragment length polymorphism (AFLP) protocol for genotyping the malarial parasite Plasmodium falciparum

Published online by Cambridge University Press:  26 November 2001

J. M. RUBIO ,
P. J. BERZOSA  and
A. BENITO
J. M. RUBIO
Affiliation:
Department of Parasitology, National Center for Microbiology, National Institute of Health Carlos III, Madrid, Spain
P. J. BERZOSA
Affiliation:
Department of Parasitology, National Center for Microbiology, National Institute of Health Carlos III, Madrid, Spain
A. BENITO
Affiliation:
Department of Parasitology, National Center for Microbiology, National Institute of Health Carlos III, Madrid, Spain
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Abstract

We have established an amplified fragment length polymorphism (AFLP) protocol for identifying anonymous polymorphic loci of the malarial parasite, Plasmodium falciparum. The method consists of the following steps (i) digestion and ligation in one reaction; (ii) selective fluorescence forward primers labelled; (iii) PCR products resolved in polyacrylamide gels using the ABIPRISMTM 377 XL DNA sequencer and, (iv) the use of GenescanTM software to size the fragments. This standardized protocol distinguished between 2 standard reference clones of P. falciparum from West African and Southeast Asian and 2 Central African isolates from patients with clinical malaria. The AFLP protocol resulted in evenly distributed and reproducible band patterns for amplified fragments ranking from 163 to 489 bp long ±0·5 S.D. The primer Tru ACA labelled with the phosphoramidite 6-carboxifluorescein (FAM-blue) was easy to interpret, with a maximum of 53 bands per clone and of 81 per isolate (mixed falciparum populations) whereas the primer Tru AG labelled with the hexachlorinated analogue (HEX-green) showed a less clear pattern of bands and reproducibility than Tru ACA.

Keywords

AFLPgenotypingmalariaPlasmodium falciparum
Type
Research Article
Information
Parasitology , Volume 123 , Issue 4 , October 2001 , pp. 331 - 336
Copyright
2001 Cambridge University Press

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