This study builds upon previous studies in further defining the appropriate use of the amplified fragment length polymorphism (AFLP) for the testing of plant DNA.
Previous studies showed that consistency in DNA profiles was obtained from different somatic tissues of a cannabis plant, that the presence of resin on leaf surfaces did not impede polymerase chain reactions (PCR), and that plants suspected of clonal propagation did have identical DNA patterns while unrelated marijuana samples had different patterns. For the current study, materials and methods are described for the reproducibility studies and for discriminating between related plant sample and street seizure samples. Results showed no difference in AFLP profiles between the duplicate samples that were processed by two trained analysts. In addition, a number of students in internship were also trained to perform this DNA testing method; most achieved success in obtaining reproducible results. Factors found to most often contribute to errors were analysts' pipetting skills, attention to protocol details, failure to mix samples prior to PCR amplification, incorrect dilutions of restriction enzymes to the recommended concentrations for AFLP, and computer errors. Appropriate training and prior molecular biology experiences were the most significant factors related to analysts' achieving reproducible DNA patterns. A test case and the enzymatic digestion studies both showed that if the starting DNA for AFLP was badly damaged, some fragments would not amplify by PCR; thus, only DNA that is of sufficient quality should be used for AFLP analysis. Overall, the results from replication and population studies show that the AFLP method is useful for any single source plant sample, since it can be applied to any organism without prior knowledge of the target segments of DNA. 3 figures and 9 references
Date Published: November 1, 2005