Simplified AFLP protocol: replacement of primer labeling by the incorporation of alpha-labeled nucleotides during PCR.

Over the last few years, PCR-based technologies have become established as systems for genetic mapping and fingerprinting in a variety of organisms (1,8,9). AFLP (7) is a recently developed multilocus marker technique that has been widely used for identifying polymorphisms in both prokaryotic and eukaryotic organisms (3,4,6). In AFLP, a subset of restriction fragments from a genomic DNA digest is selectively amplified in the PCR. The technique does not require prior knowledge of DNA sequences and has proved to be both robust and reliable because it uses stringent reaction conditions. According to the standard AFLP protocol (7), fingerprints are visualized by end-labeling one of the AFLP primers with polynucleotide kinase and γ-[32P]-dATP or γ-[33P]-dATP. A commercially available AFLP kit (AFLP Analysis System I; Life Technologies, Rockville, MD, USA) is also based on primer end-labeling. We found that the primer labeling step can be omitted when α-labeled nucleotides are incorporated into PCR products during amplification. This simplification of the protocol proves particularly useful when a large number of primers have to be screened for suitability to generate polymorphisms and/or when α-labeled nucleotides are available for other purposes. Here, we describe a protocol for setting up AFLP analysis without primer labeling and without relying on commercial kits. In the original AFLP protocol (7), genomic DNA is digested with EcoRI and MseI. We used Tru1I (MBI Fermentas, St. Leon-Rot, Germany) as an economical substitute for MseI (both enzymes cleave the sequence /AATT). To simplify the protocol, both enzymes were added at the same time, and the reaction mixture was incubated successively at 37°C (the optimal temperature for EcoRI) and at 65°C (optimum for Tru1I). To illustrate our protocol, 200 ng of DNA extracted from the tissue of Lymantria dispar (Lepidoptera, Lymantriidae) and purified using spermine precipitation (5) were restricted with 10 U EcoRI (Amersham Pharmacia Biotech, Freiburg, Germany) and 1 U Tru1I in 1× R buffer (both from MBI Fermentas) in the presence of 0.1 mg/mL BSA for 1.5 h at 37°C, followed by 1.5 h at 65°C in a total volume of 10 μL. Added to the digested DNA were 1 pM of both EcoRI and Tru1I oligonucleotide adapters (see Table 1), ligation buffer and 0.25 U T4 DNA ligase (both from US Biochemicals, Cleveland, OH, USA). The solution was incubated at 20°C for 2 h and diluted tenfold with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Similar to the original protocol, PCR was performed in two steps: (i) pre-amplification with primers corresponding to the EcoRI and Tru1I adapters and (ii) selective amplification with primers prolonged by 1 to 3 nucleotides. For the preamplification, 27 ng of EcoRI primer (5′-GACTGCGTACCAATT-3′) and 30 ng of Tru1I primer (5′-GATGAGTCCTGAGTAA-3′) were mixed with 1 μL of diluted ligated DNA, 1× AFLP buffer (10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), 0.1 mM dNTPs (Amersham Pharmacia Biotech) and 0.2 U Taq DNA polymerase (MBI Fermentas) in a total volume of 5 μL. The samples were incubated in a PTC100 thermal cycler (MJ Research, Watertown, MA, USA) for 20 cycles that consisted of 94°C for 30 s, 56°C for 1 min and 72°C for 1 min. After the preamplification step, the PCR products were diluted 50-fold with TE buffer and further used for selective amplification. Selective amplification with the incorporation of α-[33P]-dATP was carried out in a total reaction volume of 5 μL containing 1.25 μL diluted, pre-amplified template DNA, 1× AFLP buffer, 10 μM dATP, 0.1 mM each of dCTP, dGTP and dTTP, 0.2 U Taq DNA polymerase, 2.7 ng and 7.6 ng of selective EcoRI and Tru1I primers, respectively, and 0.5 μCi α-[33P]-dATP (3000 Ci/mM; Amersham Buchler, Braunschweig, Germany). The PCR temperature profile began with one cycle at 94°C for 30 s, 65°C for 30 s and 72°C for 1 min. The annealing temperature was then lowered by 0.7°C for each of 12 cycles, followed by 23 cycles at 94°C for 30 s, 56°C for 1 min and 72°C for 1 min. After amplification, the samples were denatured by adding an equal volume (5 μL) of formamide buffer (97.95% formamide, 0.025% xylene cyanol FF, 0.025% bromophenol blue and 2% of 0.5 M EDTA, pH 8.0) and heating for 3 min at 80°C. Four microliters of the samples were loaded on a 5% denaturing polyacrylamide gel and electrophoresed in 1× TBE buffer (0.1 M Tris-HCl, 0.1 mM EDTA, pH 8.0) at 50 W for 1.5 h in a 40 cm long electrophoretic chamber (Sequencing gel electrophoresis apparatus; Life Technologies). The gel was dried for 40 min at 70°C in a gel dryer (Bio-Rad Laboratories, Hercules, CA, USA), exposed to a phospho-imaging screen (Fujifilm Imaging Plate; raytest, Straubenhardt, Germany) for approximately 18 h and scanned using a Fujifilm Bioimaging Analyzer System (BAS1000); raytest. Using the above protocol with the incorporation of α-[33P]-dATP, AFLP fingerprints of insect and fungal genomes displaying large numbers of DNA polymorphisms were successfully generated (data not shown). No differences in the banding pattern were observed between products radiolabeled using end-labeled primers and those visualized by the incorporation of α-[33P]-dATP during PCR (data not shown). Our modification of the original procedure by Vos et al. (7) has several advantages: (i) incorporation labeling results in a reduction of the required Benchmarks