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Use of a single primer to fluorescently label selective amplified fragment length polymorphism reactions

2004; Future Science Ltd; Volume: 37; Issue: 6 Linguagem: Inglês

10.2144/04376bm03

1940-9818

L F Habera, Naomi R. Smith, Ryan S. Donahoo, Kurt Lamour,

Genomics and Phylogenetic Studies

BioTechniquesVol. 37, No. 6 BenchmarksOpen AccessUse of a single primer to fluorescently label selective amplified fragment length polymorphism reactionsLedare Habera, Naomi Smith, Ryan Donahoo & Kurt LamourLedare HaberaUniversity of Tennessee, Knoxville, TN, USA, Naomi SmithUniversity of Tennessee, Knoxville, TN, USA, Ryan DonahooUniversity of Tennessee, Knoxville, TN, USA & Kurt Lamour*Address correspondence to: Kurt Lamour, The University of Tennessee, Institute of Agriculture, Knoxville, TN 37996-4560, USA. e-mail: E-mail Address: klamour@utk.eduUniversity of Tennessee, Knoxville, TN, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04376BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Amplified fragment length polymorphism (AFLP) markers are used for a variety of genetic applications including population genetic studies (1,2), mapping (3), and gene discovery (4). Genomic DNA or cDNA is digested with restriction enzymes in the presence of synthetic adaptors in a "restriction/ligation" reaction that produces a population of anonymous DNA fragments with known ends. Selective primers complementary to the adaptors with additional 3′ nucleotides are then used to amplify specific subsets of the modified fragments under stringent PCR conditions. Typically, the resulting AFLP profiles are separated using polyacrylamide, and the markers are scored for presence or absence (5).One of the advantages of AFLP is that a large number of markers can be generated based on differential combinations of selective primers (5). The use of fluorescently labeled selective primers in conjunction with a capillary sequencing device provides a robust platform for resolving fragment profiles, but a limiting factor for this type of resolution is the cost of fluorescently labeled selective primers. Cost includes the relatively short lifespan of fluorescently labeled primers. We report here a strategy for labeling selective AFLP products using a single fluorescently labeled AFLP primer. This strategy requires no new technical skills on the part of the investigator and provides greater consistency as batch-to-batch variation in primer synthesis is lessened.Our AFLP protocol is very similar to the original description of AFLP by Vos et al. through the selective amplification step with the addition of a labeling step (6). Specifically, unlabeled selective reactions are diluted 500× and used as template for an additional nonselective reaction using a fluorescently labeled preselective primer. We have tested this strategy on a variety of organisms including Phytophthora capsici, horseweed (Conyza Canadensis), dogwood (Cornus florida), and soybean cyst nematodes (SCN) (Heterodera glycines) comparing AFLP profiles generated using fluorescently labeled selective primers versus AFLP profiles labeled using nonselective primers. In each case, the profiles generated were identical (Figure 1).Figure 1. Phytophthora capsici amplified fragment length polymorphism (AFLP) electropherograms generated using selective primers E + AC/M + CC.(A) The fragments were labeled using a labeled E + AC in a standard reaction. (B) The fragments were labeled using a labeled E + 00 in an additional nonselective labeling reaction. Fragments were resolved and visualized using a CEQ 8000 Genetic Analysis System. nt, nucleotides; a.u., arbitrary units.In brief, high molecular weight DNA was extracted from approximately 10 mg of freeze-dried material using DNeasy® mini-kits (Qiagen, Valencia, CA, USA). Freeze-dried starting material consisted of Phytophthora mycelium grown in antibiotic-amended V8 broth, SCN eggs, and leaf discs of horseweed and dogwood leaves. DNA was quantified on an agarose gel with known standards. EcoRI was obtained from Invitrogen (Carlsbad, CA, USA), while MseI and T4 DNA ligase were obtained from Takara (Madison, WI, USA). EcoRI and MseI adapters and primers for ligation and amplification reactions were obtained from Integrated DNA Technologies (Coralville, IA, USA). For sequences for the adapters and primers, see Vos et al. (6). The fluorescently labeled primers were obtained from Proligo (Boulder, CO, USA) and are comprised of the EcoRI core sequence with and without any selective nucleotides and either the WellRED D4-PA label (Proligo) or the WellRED D3-PA label at the 5′ end.Restriction and ligation reactions were carried out simultaneously in 11-µL volumes containing 150 ng of genomic DNA in 1× T4 ligase buffer, 0.5 M NaCl, 0.045 M bovine serum albumin (BSA), 0.5 µM EcoRI adapter, 5.0 µM MseI adapter, 5 U EcoRI, 1 U MseI, and 1 U T4 DNA Ligase. Reactions were placed on a Dyad® PTC-220 thermal cycler (MJ Research, Waltham, MA, USA) for 37°C for 2 h followed by 72°C for 15 min or allowed to incubate overnight at room temperature. Following the restriction-ligation reactions, the template was diluted by the addition of 189 µL 10 mM Tris.Preamplification reactions were carried out in 20-µL volumes containing 5.0 µL dilute restriction ligation products, 1× PCR buffer, 0.2 mM dNTPs, 0.275 µM EcoRI and MseI primers, and 1 U Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA, USA). Preamplification reactions were performed on a Dyad PTC-220 thermal cycler using the following cycling parameters: initial incubation at 72°C for 2 min, followed by 20 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, with a final extension at 72°C for 2 min, and a final incubation at 60°C for 30 min. Upon completion of the preamplification, the reaction products were diluted by adding 135 µL 10 mM Tris.Selective amplification reactions were carried out with both labeled and unlabeled selective primer pairs in 20-µL volumes containing 5.0 µL dilute preamplification products, 1× PCR buffer, 0.2 mM dNTPs, 0.275 µM EcoRI + 2 primer (E + 2), and MseI + 2 primer (M + 2) for Phytophthora and SCN, and EcoRI + 3 primer (E + 3) and MseI + 3 primer (M + 3) for horseweed and dogwood, and 1 U Taq DNA polymerase. Selective amplification reactions were performed using the following cycling parameters: initial incubation at 94°C for 2 min, followed by one cycle of 94°C for 20 s, 66°C for 30 s, and 72°C for 2 min; cycles 2–11: 94°C for 20 s, decrease 1°C every cycle for 30 s, and 72°C for 2 min; cycles 12–31: 94°C for 20 s, 56°C for 30 s, and 72°C for 2 min, followed by a final incubation at 60°C for 30 min. Upon completion of the program, the unlabeled selectively amplified products were diluted 1:500 in 10 mM Tris.For the selective reactions performed with unlabeled selective primers, a second round of selective amplification was performed using the 1:500 dilute products as template. We tested 1:10, 1:100, 1:500, 1:750, and 1:1000 dilutions, and 1:500 provided the optimal signal. This extra labeling reaction is accomplished by substituting the E + 2 or the E + 3 with an E + 00 EcoRI primer containing either the WellRED D4-PA or the WellRED D3-PA label.Fluorescent products from both the traditional and second round selective amplification were analyzed on a CEQ™ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA) using the manufacturer's protocols. Figure 1 illustrates typical results from selective AFLP reactions labeled using this single primer strategy versus AFLP reactions using fluorescently labeled selective primers. We have tested this strategy on over 100 genomic DNA samples comparing both labeling methods and have found no significant differences between the AFLP profiles generated. Co-loading of AFLP fragments generated using two different WellRED dyes (multiplexing) gave identical results to loading each product separately. The key to this procedure is the 500-fold dilution of the unlabeled selective PCR products. Considering the cost of individual dye-labeled primers and the limited lifespan of fluorescent dyes, this strategy can significantly reduce the cost of generating AFLP markers from multiple selective primer combinations.AcknowledgmentsWe are grateful to Matt Smith, Sharyce Banks, Catherine Zama, Beth Wilson, and Melinda Tierney for technical assistance. This work was supported in part by funds from the Tennessee Soybean Promotion Board.Competing Interests StatementThe authors declare no conflicts of interest.References1. Lamour, K.H. and M.K. Hausbeck. 2001. The dynamics of mefenoxam insensitivity in a recombining population of Phytophthora capsici characterized with amplified fragment length polymorphism markers. Phytopathology 91:553–557.Crossref, Medline, CAS, Google Scholar2. Lamour, K.H. and M.K. Hausbeck. 2003. Effect of crop rotation on the survival of Phytophthora capsici and sensitivity to mefenoxam. Plant Dis. 87:841–845.Crossref, Medline, CAS, Google Scholar3. Van der Lee, T., I. De Witte, A. Drenth, C. Alfonso, and F. Govers. 1997. AFLP linkage map of the oomycete Phytophthora infestans. Fungal Genet. Biol. 21:278–291.CAS, Google Scholar4. Durrant, W.E., O. Rowland, P. Piedras, K.E. Hammond-Kosack, and J.D.G. Jones. 2000. cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell 12:963–977.Crossref, Medline, CAS, Google Scholar5. Blears, M.J., S.A. De Grandis, H. Lee, and J.T. Trevors. 1998. Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications. J. Ind. Micro. Biotech. 21:99–114.Crossref, CAS, Google Scholar6. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van der Lee, M. Hornes, A. Frijters, J. Pot, et al.. 1995. AFLP: a new technique for DNA fingerprinting. 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This work was supported in part by funds from the Tennessee Soybean Promotion Board.Competing Interests StatementThe authors declare no conflicts of interest.PDF download