Modification of the AFLP Protocol Applied to Honey Bee ( Apis mellifera L.) DNA

Abstract

The established amplified fragment-length polymorphism (AFLP) protocol was simplified and optimized for honey bee DNA (Apis mellifera L.). Compared to the original method, the following simplifications were made: (i) the digestion of DNA and ligation of the adapters are performed in one reaction vs. two, (ii) one restriction enzyme is used vs. two and (iii) amplification is accomplished in one reaction vs. two. PCR products are resolved in agarose-Synergel instead of polyacrylamide and are visualized by ethidium bromide staining rather than by autoradiography of labeled primers. Using the modified procedure for honey bee DNA, high reproducibility of the band patterns of PCR products and low sensitivity to the amplification conditions were seen. Analysis of honey bee DNA revealed considerable genetic variability within and between African and European bee samples. African- and European-specific fragments were found.

Genetic diversity of Hirsutella thompsonii isolates from Thailand based on AFLP analysis and partial beta-tubulin gene sequences

“…For each sample, 0.3-2.0 mg of genomic DNA was digested with EcoRI and ligated to EcoRI adaptors in a single overnight reaction at 37°C (Suazo and Hall, 1999). The digestion-ligation reactions were diluted with TE buffer to a final volume of 200 mL and stored at -20°C.…”

Genomic diversity of Astragalus cicer microsymbionts revealed by AFLP fingerprinting

“…Lanes: 1, 47-"Gene Ruler TM 100 bp DNA Ladder" Fermentas (100-3,000 bp); 2-AZ1; 3-AZ2; 4-AZ3; 5-AZ5; 6-AZ6; 7-AZ7; 8-AZ8; 9-AW1/2; 10-AW1/3; 11-AW1/5; 12-AW1/8; 13-AW1/14; 14-ACMP1; Pst-GC, and Pst-A was chosen. The AFLP technique applied for DNA of any origin and complexity uses arbitrary sequences to generate polymorphic amplicons from DNA digested by restriction enzymes (Blears et al, 1998;Lan and Reeves, 2000;Lin et al, 1996;Ranamukhaarachchi et al, 2000;Savelkoul et al, 1999;Suazo and Hall, 1999). It combines RAPD simplicity with RFLP reliability without the need for expensive equipment and knowledge of the DNA sequences.…”

Application of the AFLP method to differentiate Genista tinctoria microsymbionts

“…Since the original publication of the AFLP procedure by Vos et al in 1995, some modifications of this method, based on one restriction enzyme and a single adaptor, have been described (Suazo and Hall, 1999;Tyrka et al, 2002).…”

“…The sequences of the primers applied for PCR reaction define the number of the restriction fragments that are to be amplified. Primers fully complementary to the adaptors (nonselective primers) amplify indiscriminately all "tagged" fragments, whereas primers with one to three arbitrary nucleotides (selective primers), amplify only part of them.Since the original publication of the AFLP procedure by Vos et al in 1995, some modifications of this method, based on one restriction enzyme and a single adaptor, have been described (Suazo and Hall, 1999;Tyrka et al, 2002).In this study, simplified AFLP analysis with one endonuclease PstI and two selective primers was used for typing of G. tinctoria microsymbionts and for assessing their genomic diversity. …”