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AFLP technology for DNA fingerprinting
Author: Vuylsteke, M.
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"AFLP technology for DNA fingerprinting Marnik Vuylsteke 1,2 , Johan D Peleman 3 & Michiel JT van Eijk 3 1 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Ghent, Belgium. 2 Department of Molecular Genetics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium. 3 Keygene N.V., Agro Business Park 90, NL-6708 PW Wageningen, The Netherlands. Correspondence should be addressed to M.V. (marnik.vuylsteke@psb.ugent.be). Published online 31 May 2007; corrected online 14 August 2008 (details online); doi:10.1038/nprot.2007.175 The AFLP technique is a powerful DNA fingerprinting technology applicable to any organism without the need for prior sequence knowledge. The protocol involves the selective PCR amplification of restriction fragments of a total digest of genomic DNA, typically obtained with a mix of two restriction enzymes. Two limited sets of AFLP primers are sufficient to generate a large number of different primer combinations (PCs), each of which will yield unique fingerprints. Visualization of AFLP fingerprints after gel electrophoresis of AFLP products is described using either a conventional autoradiography platform or an automated LI-COR system. The AFLP technology has been used predominantly for assessing the degree of variability among plant cultivars, establishing linkage groups in crosses and saturating genomic regions with markers for gene landing efforts. AFLP fragments may also be used as physical markers to determine the overlap and positions of genomic clones and to integrate genetic and physical maps. Crucial characteristics of the AFLP technology are its robustness, reliability and quantitative nature. This latter feature has been exploited for co-dominant scoring of AFLP markers in sample collections such as F 2 or back-cross populations using appropriate AFLP scoring software. This protocol can be completed in 2?3 d. INTRODUCTION The AFLP (amplified fragment length polymorphism) method is a DNA fingerprinting technique based on selective PCR amplifica- tion of restriction fragments from a total digest of genomic DNA of any origin or complexity such as prokaryotes, plants, animals and human 1 . The AFLP technique was originally conceived for the construction of high-density linkage maps for application in positional cloning of genes and molecular breeding. Because the AFLP technology is essentially suited for fingerprinting and map- ping of any genomic DNA, it is equally suited for applications in genetic analysis such as genetic relationship and diversity assess- ments (e.g., plants 2 ,bacteria 3 ), establishment of ?essential deriva- tion? among plant varieties 4,5 and association studies in natural 6 and breeding populations 7?9 . In addition, the AFLP technology is applicable for genome research, such as characterizing the level and target sites of cytosine methylation 10,11 , fingerprinting and identi- fying overlapping clones in the construction of high-resolution BAC (bacterial artificial chromosome) physical maps 12 ,forinte- grating physical and genetic maps 13,14 and for high-throughput enrichment of radiation hybrid maps 15 . AFLP markers offer several advantages over other currently used DNA markers, such as simple sequence repeats and single nucleo- tide polymorphisms. Foremost among these is that the AFLP technology requires no prior sequence information and, hence, has a relatively low start-up cost. In addition, the AFLP technique is very amenable to automation and is highly multiplexed, which offers the potential to improve the efficiency and to increase the throughput of marker data production in organisms that lack the genomics platform necessary to allow the development of genotyping microarrays. The AFLP technique also has a number of limitations. In case of low (less than 90%) overall sequence homologies between samples, AFLP fingerprints will share very few common fragments. It has indeed been shown in bacteria that relationships at the subspecies level may not be detected by the AFLP technique 3 . The AFLP method cannot, therefore, be used for comparative genome analysis. Detection of markers in genomic DNA with very little sequence variation may be poor despite the large numbers of fragments that can be tested for polymorphisms. In these cases, marker systems that combine AFLP with micro- satellites 16 or transposons 17 may be superior. The first step of the AFLP procedure (see Fig. 1) involves the preparation of templates by restriction digestion of DNA, typically p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h Gel electrophoresis Total genomic DNA Restriction digestion Adapter ligation Preamplification Selective amplification (1) (2) (3) (4) (5) NNN NN N N Figure 1 | Outline of the AFLP procedure. Template fragments are generated by: (1) digestion of genomic DNA with a combination of the two restriction enzymes EcoRI and MseI (blue and red arrows represent EcoRI and MseI restriction enzyme sites, respectively); (2) ligation of the double-stranded EcoRI- (blue) and MseI- (red) specific adapters to the fragment ends; (3) a pre-amplification step using primers that match the adapter sequences and that carry each one selective nucleotide (represented by N) at their 3 end are used to PCR-amplify subsets of the EcoRI/MseI templates; (4) a final selective PCR-amplification step in which additional selective nucleotides are added to the EcoRI and MseI primers; and (5) the electrophoretic size fractionation and the display on denaturing polyacrylamide gels of the EcoRI/MseIamplificationproducts. NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1387 PROTOCOL with two different restriction enzymes. The two restriction enzymes used are generally (but not necessarily) a rare cutter and a frequent cutter. The frequent cutter is used to generate fragments that are in the 50?500 bp length range resolvable by electrophoresis. The rare cutter is used to limit the number of fragments that can be amplified and, hence, to define the number of effective AFLP amplicons. Subsequently, double-stranded adapters are ligated to the ends of the restriction fragments. The second step of the AFLP procedure is the PCR amplification of subsets of restriction fragments using selective AFLP primers. The common parts of these primers correspond to the adapter and restriction enzyme recognition sequences, and they have a number of additional bases at the 3�-end extending into the restriction fragments, called the selective nucleotides (see Fig. 2). These selective nucleotides ensure that only a subset of restriction fragments is amplified to a detectable level, that is, those fragments where the nucleotides flanking the restriction site match the primer extensions. AFLP fingerprinting of low-complexity DNA (plasmids, cosmids and BACs) requires no selective nucleotides. For small genome-sized (5?100 Mb) organisms such as bacteria and fungi, up to two selective nucleotides for each of the AFLP primers are commonly required. AFLP fingerprinting of more complex, large genomes (greater than 100 Mb) is usually carried out with two or three selective bases in one or both primers and is generally performed in two consecutive steps: a pre-amplification step, reducing the complexity of the template mixture, and a final selective step. Detection of the AFLP fragments is made possible by radio- active or fluorescent labeling of one of the two AFLP primers used in the final selective amplification reaction. The final step of the AFLP technique is the electrophoretic size fractionation of the fingerprints. For this purpose the labeled reaction products are separated on denaturing polyacrylamide gels similar to those used for sequencing. In the case of conventional gel electrophoresis using radio-labeled primers, gels are either dried on paper or fixed on glass plates after electrophoresis, and AFLP images may be generated using either conventional autoradiography or phosphor- imaging technology (Fig. 3a). In the case of gel electrophoresis using infrared dye (IRD) or fluorescently labeled primers, AFLP images may be generated using LI-COR (see Fig. 4) or Applied Biosystems (ABI) and MegaBACE automated DNA sequencers, respectively. A crucial characteristic of the AFLP technology is the quantita- tive nature of the competitive PCR amplification of restriction fragments, which is based on the kinetics of the AFLP amplification reaction. Specifically, a single pair of PCR primers is used to amplify a subset of restriction fragments simultaneously, with each parti- cular fragment from the subset competing for the same primers according to its initial relative abundance. These competitive amplification kinetics remain for as long as effective amplification can be sustained in the reaction mixture. Owing to this feature, the relative intensity of a band in an AFLP fingerprint pattern reflects the original abundance of that fragment in the AFLP template, such that band intensities can be compared among samples 18,19 as long as loading differences between lanes are accounted for. Because genomic polymorphisms manifest themselves predominantly as single-base mutations that affect either the restriction site or the selective bases immediately adjacent to them, such polymorphisms result in dominant PCR phenotypes; that is, the presence of a mutation causes the loss of a fragment from a fingerprint. In the case of a heterozygous mutation, however, the difference between 2n and 1n can be clearly distinguished from the band intensities, which reflect PCR product concentrations (100 and 50%, respec- tively). This quantitative nature of the AFLP technology has been exploited widely for routine co-dominant scoring of AFLP markers in segregating populations (see below for further discus- sion), establishing that the technology can clearly be used to discriminate homozygous from heterozygous loci in the majority of cases 20 . The AFLP protocol described below and illustrated in Figure 1 details all steps in the AFLP procedure, except the isolation of total DNA, as follows: (i) the preparation of template fragments by digestion of the genomic DNA with a combination of the two restriction enzymes EcoRI and MseI; (ii) the ligation of double- stranded adapters to the fragment ends; (iii) a first selective PCR amplification, also called pre-amplification, of EcoRI/MseIfrag- ments with a combination of an EcoRI and an MseI primer with one selective base each; (iv) a final selective amplification step in which additional selective nucleotides are used in the EcoRI and MseI primers; (v) the electrophoretic analysis of the 50?100 EcoRI/MseI amplification products on standard denaturing poly- acrylamide gels and detection of AFLP fragments using either conventional gel electrophoresis, radio-labeled primers and auto- radiography, or LI-COR automated DNA sequencers and IRD detection technology. The two detection options are virtually identical through the preparation of template fragments, except in the final selective amplification step, where IRD-labeled primers are substituted for the radioactively labeled primers when using the LI-COR automated DNA sequencer. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h EcoRI-adapter 5?-CTCGTAGACTGCGTACC AATTCGAC-internal sequence-3? 3?-CATCTGACGCATGG-TTAAGCTG-internal sequence-5? PstI-adapter 5?-CTCGTAGACTGCGTACA-TGCAGGAC-internal sequence-3? 3?-CATCTGACGCATGT ACGTCCTC-internal sequence-5? MseI-adapter 5?-GACGATGAGTCCTGAG TAAGAC-internal sequence-3? 3?-TACTCAGGACTC-ATTCTG-internal sequence-5? EcoRI-primer 5?-GACTGCGTACCAATTCNNN-3? PstI-primer 5?-GACTGCGTACATGCAGNNN-3? MseI-primer+0 5?-GATGAGTCCTGAGTAA-3? MseI-primer+1 5?-GATGAGTCCTGAGTAAG-3? MseI-primer+2 5?-GATGAGTCCTGAGTAAGA-3? Figure 2 | Schematic for adapter and primer design for the two rare cutters EcoRI and PstI and the frequent cutter MseI. Adapters consist of a core sequence (black) and an enzyme-specific sequence (red). The enzyme-specific sequence allows the ligation of the adapters to the resulting restriction fragments (green) without restoring the original restriction sites. In this way, ligated adapters create a target site for the AFLP primers in the subsequent amplification reactions. For this purpose, primer design matches the core (black), the enzyme-specific (red) and the restriction-site remnant (green) sequence. Primers may have one or a number of additional bases at the 3-end extending into the restriction fragments, called the selective nucleotides (represented by N, in blue). AFLP primers are named ?+0? when they have no selective bases (only the core, enzyme-specific and restriction-site remnant sequence),?+1? when they have a single selective base,?+2? when they have two selective bases, and so on. Adapters and primers for other restriction enzymes are similar to these but have enzyme-specific parts corresponding to the respective enzymes. 1388 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL Experimental design Preparation and quality assessment of genomic DNA. Complete restriction is crucial for good quality of AFLP fingerprints. DNA preparations, therefore, need to be of sufficient quality to allow complete digestion by the restriction enzymes. A minimum of 100 ng of eukaryotic DNA is recommended for template generation. DNA concentrations can be determined by measuring OD 260 , but we recommend running a small aliquot of DNA on a 1% agarose gel, next to a series of phage-l DNA dilutions ranging from 50 to 500 ng. The gel image will also allow inspection of the integrity of the original DNA. Substantial smearing below the main band of high molecular weight DNA may be detrimental for AFLP fingerprinting quality. Choice of restriction enzymes. The protocol describes the gene- ration of templates for AFLP reactions by the restriction of the DNA with the restriction enzyme combination (EC) EcoRI/MseI, which is one of the most commonly used in plant species and micro-organisms (for the latter see, e.g., AFLP analysis system for micro-organisms; http://www.invitrogen.com). Obviously, altering the EC requires appropriate design of adapter and amplification primers and adaptation of the digestion conditions. The selection of the appropriate ECs is determined by the efficiency of poly- morphism detection, the genome coverage and AFLP marker distribution. Genome coverage and AFLP marker distribution are mainly determined by the AT-content of the DNA. For AT-rich genomes, including most eukaryotic DNAs, AT-rich restriction enzymes such as MseI (recognition sequence TTAA) and EcoRI (recognition sequence GAATTC) will generally produce good genome coverage and fragments that are in the optimal size range for both PCR amplification and separation on denaturing polyacrylamide gels. For CG-rich genomes, CG-rich restriction enzymes such as PstI (recognition sequence CAGCTG) and TaqI (recognition site TCGA) are more appropriate restriction enzymes. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h M034 M033 M032 M031 M030 M029 M028 M027 M026 M025 M024 M023 M022 M021 M020 M019 M018 M017 M016 M015 M014 M013 M012 M011 M010 M009 M008 M007 M006 M005 M004 M003 M002 M001 34...4321 M001 M002 M003 M004 M005 M006 M007 M008 M009 M010 M011 M012 M013 M014 M015 M016 M017 M018 M019 M020 M021 M022 M023 M024 M025 M026 M027 M028 M029 M030 M031 M032 M033 M034 ab ...434321 Figure 3 | AFLP analysis of 32 tomato F 2 segregants. (a) Gel image of the AFLP analysis of 32 tomato F 2 segregants (in lanes 3?34) and their parental lines (in lanes 1 and 2) using a PstI+ AT/MseI + CTG primer combination. Selective amplification was performed using a [g- 33 P]ATP- labeled PstI+AA primer and fingerprints were visualized using phosphorimaging technology. Thirty-four AFLP markers segregating in this F 2 population were co-dominantly scored using AFLP-QuantarPro software. The goal of co-dominant scoring of AFLP markers is unequivocally to distinguish homozygous from heterozygous genotypes based on quantitative measurements of the band intensities. This allows extraction of more genetic information from AFLP fingerprints than dominant (presence/absence) scoring. For homozygous individuals (2n), the band intensity is expected to be twice that for heterozygous individuals (1n). Thus, if band intensities can be measured quantitatively, the difference between 2n (100%) and 1n (50%) for an AFLP locus can be scored. In this example, 34 co-dominant AFLP markers are indicated, labeled M001-M0034 on the left-hand side. (b) AFLP dataset resulting from the AFLP analysis shown in (a). The dataset consists of 34 AFLP markers (labeled M001-M034 on the left hand side) which were co-dominantly scored using AFLP-QuantarPro software in the 32 F 2 individuals (labeled 3-34 across the top) and their parental lines(labeled1-2acrossthetop).Inthecaseofan F 2 population, individuals? marker genotypes may be assigned to one of the three genotype classes (A: homozygous absent; H: heterozygous; B: homozygous present). If heterozygous genotypes can not unequivocally be distinguished from homozygous B genotypes, a C genotypic score (i.e. not genotype A) will be assigned. If heterozygous genotypes can be distinguished from homozygous A genotypes, a D genotypic score (i.e. not genotype B) will be assigned. Such AFLP datasets typically serve as a starting point for further genetic analysis. NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1389 PROTOCOL The efficiency of polymorphism detection by AFLP can be adjusted by targeting CpG dinucleotide motifs in mammals (e.g., by the use of TaqI) or CpNpG trinucleotide motifs (e.g., by the use of PstI) in plants. Since such motifs are prone to an increased level of mutation, an increased level of polymorphism detection is observed. For example, in plants (e.g., maize and sor- ghum) sets of PstI/MseI primer combina- tions (PCs) have a significantly higher polymorphism information content than sets of EcoRI/MseIPCs 11,21 . In farm animal species (chicken 22 ,cattle 23 and pig 24 ), the EC of EcoRI/TaqI is most commonly used. However, other ECs have been used in farm animal species as well, such as EcoRI/HinPI and EcoRI/MspI in chicken 25 and EcoRI/HinPI in turkey 26 . For parasites, such as Oesophagostomum, evaluation of different ECs demonstrated that the use of HindIII/BglII, a combination of two six cutters, was the most effective to investigate genetic diversity by the AFLP method 27 . Te mp la te pr ep ar at io n. Adapters consist of a core sequence and a restriction enzyme? specificsequence(Fig. 2) and are prepared by adding equimolar amounts of both strands. Adapters are not phosphorylated, to prevent adapter?adapter ligation. The sequence allows the ligation of the adapters to he resulting restriction fragments with- out restoring the original restriction sites. Ligated adapters create a target site for the AFLP primers in the subsequent amplifica- tion reactions. Primer design and preparation. For selec- tive amplification of subsets of AFLP tem- plates, primers are used that correspond to the core and the enzyme-specific sequence of the adapter and to the remnant sequence of the restriction site (Fig. 2). They have one or a number of (up to three) additional bases at the 3�-end extending into the restriction fragments, called the selective nucleotides (Fig. 2). AFLP primers are named ?+0? when they have no selective bases (only the core and enzyme-specific sequence), ?+1? when they have a single selective base, ?+2? when they have two selective bases, and so on. Only one of the two selective AFLP primers used is labeled in the final selective amplifica- tion step because the mobilities of the two strands of a DNA fragment on sequencing gels are generally slightly different, resulting in a lower resolution of the PCR product on the gel. There is no preference which primer to label, but generally the primer corresponding to the rare cutter is chosen. Selective amplification of AFLP fragments obtained with other restriction ECs than detailed here can be performedusingessentialythesameprotocolwithappropriate primers and adapters. Choice of radioactive versus fluorescent detection systems. LI- COR and ABI have adapted the AFLP technique for use with IRD or fluorescent dye detection technology, respectively. Detection of p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h > Bi-allelic marker > Bi-allelic marker Mono-allelic marker Mono-allelic marker 58...4321 MW 500 bp 400 bp 300 bp 200 bp 100 bp Figure 4 | Gel image of an AFLP analysis of 56 Arabidopsis Recombinant Inbred Line (RIL) segregants analyzed using an EcoRI + AA/MseI + CAA primer combination. RILs are shown in lanes 3?58 and their parental lines in lanes 1 and 2. A 10-bp molecular weight (MW) marker was included on the left of the gel image. Selective amplification was performed using an infrared dye 700?labeled EcoRI + AA primer, and digital images of the fingerprints were obtained from the LI-COR automated sequencer. Because single- base mutations affect either the restriction site or the selection bases immediately adjacent to them, the presence of such a mutation causes the loss of an AFLP fragment from a fingerprint and results in a mono- allelic AFLP marker segregating present/absent in a RIL mapping population. An insertion/deletion in the sequence of one of the two AFLP fragment alleles causes a size difference between the two AFLP marker alleles, resulting in two AFLP markers showing a complementary present/absent segregation pattern. It is clear from this gel image that bi-allelic AFLP markers are identified with a much lower frequency than mono-allelic AFLP markers. Two examples of each type of AFLP marker are pointed out on the figure. 1390 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL AFLP fragments using IRD or fluorescent dye detection technology offers several advantages over conventional detection using radio- labeled primers and autoradiography: the use of radioactivity is eliminated, the cost of dye-labeled primers is less than the cost of corresponding amounts of radionucleotides for radiolabeling pri- mers and images are obtained in several hours rather than 1?3 d. In addition to the safety and convenience benefits of AFLP analysis on automated sequencers, the throughput of the AFLP analysis can be doubled by running multiplexed AFLP reactions on two-dye LI-COR 28 and tripled by detection using ABI or MegaBACE automated sequencers. Furthermore, owing to the larger separation power of capillary electrophoresis instruments, larger numbers of AFLP markers can be scored using these systems, specifically those in the 500?800-bp size range, which increases efficiency further. This protocol does not describe the automated AFLP analysis using ABI or MegaBACE automated sequencers and fluorescent dye detection technology. For more information on AFLP microbial and plant fingerprinting using the ABI DNA automated sequencers, we refer the reader to http://docs.appliedbiosystems.com/pebio- docs/00402977.pdf and http://docs.appliedbiosystems.com/pebio- docs/04303146.pdf, respectively. Factors affecting co-dominant scoring of AFLP markers. Various factors affect the proportion of AFLP markers that can be co- dominantly scored based on fragment intensity levels. One of the main factors in this respect is the genetic relatedness of individual samples included in the co-dominant scoring process. Specifically, it is commonly observed that high proportions of co-dominantly scored AFLP markers are usually reached in geneti- cally less complex sample collections such as F 2 or back-cross populations, and lower percentages are observed when screen- ing large germplasm collections. Second, the ploidy level of the organisms is important; that is, co-dominant scoring is easier in diploid organisms than in tetra- or hexaploid organisms. Third, DNA quality is important with respect to co-dominant scoring, as increased background signal levels (probably caused by incomplete restriction/ligation) adversely affect fragment quantifi- cation. Fourth, fixing gels on the glass plate results in a better resolution and, hence, in a more accurate estimation of the band intensities. Finally, appropriate AFLP scoring software such as AFLP-QuantarPro (http://www.keygene-products.com) is essential for accurate fragment quantification and, hence, co-dominant scoring. MATERIALS REAGENTS . Tris (Biosolve, cat. no. 20092391) ! CAUTION Irritating to eyes and skin. Wear suitable protective clothing. Avoid contact with skin and eyes. Do not breath dust. . Tris-buffers (see REAGENT SETUP) . EDTA disodium salt:dihydrate (M r � 372.2 g mol C01 ; Duchefa biochemie, cat. no. E0511.1000) ! CAUTION Irritating to eyes. Avoid contact with the eyes. Wear suitable protective clothing. . SYBR Safe DNA stain in DMSO solution (Invitrogen, cat. no. S33102) ! CAUTION DMSO is irritating to eyes and skin. Avoid contact with the eyes. Wear suitable protective clothing. . Ethidium bromide (EtBr; Merck, cat. no. 1.11608.0030) ! CAUTION Harmful; possible risk of irreversible effects. Wear suitable protective clothing and gloves. . EcoRI (New England Biolabs, cat. no. R0101S) . MseI (New England BioLabs, cat. no. R0525S) . Acetic acid (HAc; Merck, cat. no. 1.00062.1000) ! CAUTION Corrosive; flammable; causes severe burns. Do not breathe vapor. Wear suitable protective clothing, gloves and eye/face protection. . Magnesium acetate (MgAc; Merck, cat. no. 1.05819.0250) (see REAGENT SETUP) . Potassium acetate (KAc; Merck, cat. no. 1.04820.1000) (see REAGENT SETUP) . Sodium acetate (NaAc; Merck, cat. no. 1.06268.250) . DTT (Immunosource, cat. no. 502A) ! CAUTION Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Do not breathe dust. Wear suitable protective clothing. . BSA (New England BioLabs, cat. no. B9001S) . MseI-F (Invitrogen) 5�-GACGATGAGTCCTGAG-3� . MseI-R (Invitrogen) 5�-TACTCAGGACTCAT-3� . MseI-adapter (see REAGENT SETUP) . EcoRI-F (Invitrogen) 5�-CTCGTAGACTGCGTACC-3� . EcoRI-R (Invitrogen) 5�-AATTGGTACGCAGTCTAC-3� . EcoRI-adapter (see REAGENT SETUP) . RL buffer (see REAGENT SETUP) . T 10 E 0.1 buffer (see REAGENT SETUP) . T4 buffer (see REAGENT SETUP) . IRD 700-labeled selective EcoRI primers for product detection using an automated LI-COR system (Biolegio) . Selective MseI primers (Invitrogen) 5�-GATGAGTCCTGAGTAAN 1?3 -3�, where N represents the selective nucleotides . Selective EcoRI primers (Invitrogen) 5�-GACTGCGTACCAATTCN 1?3 -3�, where N represents the selective nucleotides . T4 DNA-ligase (Invitrogen, cat. no. 15224-017) . ATP 100 mM solution, 25 mmol (GE Healthcare, cat. no. 27-2056-01) . Hydrochloric acid (HCl; Merck, cat. no. 1.00317.1000) ! CAUTION Corrosive; causes burns. Irritating to respiratory system. Wear suitable protective clothing, gloves and eye/face protection. . AmpliTaq DNA polymerase with PCR buffer 10C2 and MgCl 2 (25 mM) (ABI, cat. no. N8080153) . dNTP set (dATP, dCTP, dGTP, dTTP), 100 mM solutions, 4 C2 25 mmol (GE Healthcare, cat. no. 27-2035-01) . [g- 33 P]ATP (GE Healthcare, cat. no. BF1000-8MCI) ! CAUTION May cause cancer. May cause heritable genetic damage. Also harmful by contact with skin and if swallowed. Avoid exposure-obtain special instruction before use. Wear suitable protective clothing. . T4 polynucleotide kinase (New England Biolabs, cat. no. M0201S) . Spermidine-3HCl (Sigma-Aldrich, cat. no. S2501) ! CAUTION Very toxic by inhalation. Irritating to the eyes, and to the skin. Wear suitable protective clothing, gloves and eye/face protection. . Formamide (Sigma-Aldrich, cat. no. 47670) ! CAUTION May cause cancer. May cause harm to the unborn child. May cause long-term adverse effects in the aquatic environment. Avoid exposure-obtain special instruction before use. . Bromophenol blue (Merck, cat. no. 1.08122.0005) ! CAUTION Irritating to eyes; avoid contact with skin. Wear suitable protective clothing. . Xylene cyanol (Merck, cat. No. 1.10590.0005) . Glycerol (Merck, cat. no. 1.04094.1000) . 3-Methacryloxypropyltrimethoxysilane (Bind-Silane; Serva, cat. no. 28739.01) (see REAGENT SETUP) ! CAUTION Irritating to eyes, respiratory system and skin. Wear suitable protective clothing. . Acrylamide/bis-acrylamide (AabAA) 19:1 ready-made 40% mix solution (Biosolve, cat. no. 01352335) ! CAUTION May cause cancer. May cause heritable genetic damage. Harmful by inhalation and in contact with skin. Also toxic if swallowed. Irritating to eyes and skin. Avoid exposure?obtain special instruction before use. Wear suitable protective clothing. . Urea (USB, cat. no. 75826) . Boric acid (Merck, cat. no. 1.00165.1000) . Ammonium persulfate (APS; Sigma-Aldrich, cat. no. A9164) (see REAGENT SETUP) ! CAUTION Oxidizing; harmful if swallowed. Toxic in contact with skin. Very toxic by inhalation. Irritating to eyes; may cause sensitization by inhalation and skin contact. Do not breathe dust. Avoid contact with skin. Wear suitable gloves. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1391 PROTOCOL . N,N,N�,N�-tetramethylethane-1,2-diamine (TEMED; Merck, cat. no. 1.10732.0100) ! CAUTION Highly flammable, corrosive, harmful by inhalation and if swallowed. Causes burns. Wear suitable protective clothing, gloves and eye/face protection. m CRITICAL Store in the dark and keep bottle closed. . AG501-X8 mixed-bed resin (BioRad, cat. no. 142-6424) . Long Ranger stock solution 50% (Cambrex Bio Science Rockland, cat. no. 50611E) (see REAGENT SETUP) ! CAUTION May cause cancer. May cause heritable genetic damage. Harmful by inhalation and in contact with skin. Also toxic if swallowed. Irritating to eyes and skin. Avoid exposure?obtain special instruction before use. Wear suitable protective clothing. . Ultrapure 10C2 TBE buffer (1.0 M Tris, 0.9 M boric acid, 0.01 M EDTA) (Invitrogen, cat. no. 15581-044) . SmartLadder SF (Eurogentec, cat. no. MW-1800-04) . Repel-Silane ES (GE Healthcare, cat. no. 17-1332-01), 2% ready-made solution of dimethyldichlorosilane dissolved in octamethylcyclo-octosilane ! CAUTION Possible risk of impaired fertility. May cause long-term adverse effects in the aquatic environment. Wear suitable protective clothing and gloves. . Maxam 10C2 (see REAGENT SETUP) . Agarose gel (see REAGENT SETUP) . TAE running buffer (see REAGENT SETUP) EQUIPMENT . ICycler thermal cycler (BioRad) . Thermomixer comfort (Eppendorf) . Glass plates (BioRad) . SequiGenGT 38 C2 50 cm gel apparatus (BioRad) . PowerPac 3000 (BioRad) . Phosphorimager analysis system (Fuji Bas-2000 or GE-Healthcare 445 SI) . Imaging plates (Fuji or GE-Healthcare) . LI-COR long read-IR 2 4200 (LI-COR Biosciences) . Gel apparatus set (25 cm) (LI-COR Biosciences) . Whatman pure cellulose blotting sheets (3 MM Chr) 35 C2 43 cm 2 (Schleicher & Schuell BioScience, cat. no. 3030-347) . Heto dry GD-I (Heto Lab Equipment Denmark, manufactured by Hoefer Scientific instruments) REAGENT SETUP Tris-buffers [1 M Tris?HCl (pH 8.0), 1 M Tris?HCl (pH 7.5) and 1 M Tris? HAc (pH 7.5)] Dissolve 12.1 g Tris in approximately 80 ml water. Add concentrated HCl or HAc (depending on the buffer) a little at a time to reach desired pH. Finally, add water to 100 ml and autoclave. m CRITICAL Make sure buffer is at room temperature (20?22 1C) before making final pH adjustments, as the pH of Tris-buffers changes with increasing temperature. Store for up to 6 months at room temperature. 1MMgAc Dissolve 2.145 g MgAc in water. Add water to 10 ml. Filter-sterilize. Store for up to 6 months at room temperature. 4MKAc Dissolve 3.926 g KAc in water. Add water to 10 ml.mCRITICAL Store for up to 6 months at C020 1C. 1MMgCl 2 Dissolve 2.033 g MgCl 2 in water. Add water to 10 ml. Filter- sterilize. Store for up to 6 months at room temperature. 0.5 M EDTA (pH 8.0) Dissolve approximately 9 g NaOH in 400 ml water. Add 93.05 g EDTA and stir over low heat on stir plate until dissolved. m CRITICAL EDTA does not dissolve at pH less than 7.0. Add NaOH pellets to reach pH 8.0. Addwaterto500mlandautoclave.Storeforupto6monthsatroomtemperature. dNTP 5 mM of each dNTP (dATP, dGTP, dCTP, dTTP) m CRITICAL Store in aliquots (up to 2 ml) at C020 1C for up to 6 months. EcoRI-adapter (5 pmol ll C01 ) 5 ml EcoRI-F(100 mM), 5 ml EcoRI-R (100 mM), 90 mlH 2 O. Store for up to 6 months at C020 1C. MseI-adapter (50 pmol ll C01 ) 25 ml MseI-F (100 mM), 25 ml MseI-R (100 mM). Store for up to 6 months at C020 1C. RL buffer 103 Mix 1 ml 1 M Tris-HAc (pH 7.5) with 1 ml 1 M MgAc, 1.25 ml 4 M KAc, 0.077 g DTT, 50 ng ml C01 BSA (optional). Add water to 10 ml. m CRITICAL Store in aliquots (up to 2 ml) at C020 1C for up to 6 months. T 10 E 0.1 buffer Mix 1 ml 1 M Tris-HCl (pH 8.0) with 20 ml0.5MEDTA (pH 8.0). Add water to 100 ml. Store for up to 6 months at room temperature. T4 buffer 103 Mix 2.5 ml 1 M Tris-HCl (pH 7.5) with 1 ml 1 M MgCl 2 , 0.077 g DTT and 0.013 g spermidine-3HCl. Add water to 10 ml. m CRITICAL Store in aliquots (up to 2 ml) at C020 1C for up to 6 months. APS 10% Dissolve 1 g APS in water and adjust to a final volume of 10 ml. m CRITICAL The APS solution must be freshly made. Bind-Silane solution Add 30 ml HAc and 30 ml Bind-Silane to 10 ml ethanol. mCRITICALThe Bind-Silane solution must be freshly made immediately before use. Maxam 103 Dissolve 309 g boric acid and 605 g Tris in water and adjust to a final volume of 5 l. Store for up to 6 months at room temperature. Running buffer for LI-COR Dilute Ultrapure 10C2 TBE buffer tenfold. Must be freshly made. 103TAE running buffer for agarose gels Dissolve 48.4 g Tris in 250 ml water and add 11.4 ml HAc and 20 ml 0.5 M EDTA (pH 8.0). Adjust to a final volume of 1 l. Store for up to 6 months at room temperature. 1% agarose gel Add 1 g agarose to 100 ml 0.5C2 TAE running buffer. Heat in a microwave oven until completely melted. Most commonly, EtBr is added to the gel (final concentration 0.5 mgml C01 ) at this point to facilitate visualization of DNA after electrophoresis. After the solution is cooled to approximately 60 1C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. 4.5% Denaturing polyacrylamide gel solution 103 Mix 450 g urea and 112.5 ml AAbAA 19:1 40% stock solution. Add water to a final volume of 700 ml. Stir the solution at 60 1C and filter. Add 100 ml Maxam 10C2 and 4 ml EDTA 0.5 M. Add water to a final volume of 1,000 ml. m CRITICAL Store the gel solution at 4 1C in the dark for up to 30 d. 6% Long Ranger gel solution (7 M urea/1.23 TBE) Mix 3 ml Long Ranger stock solution (50%), 10.5 g urea, 3 ml 10C2 TBE buffer and 11 ml water. mCRITICAL Do not prepare and store pre-mix solutions made from 50% Long Ranger gel solution. Formamide loading dye for radioactive gels Mix 98 ml formamide, 2 ml 10 mM 0.5 M EDTA (pH 8.0), 0.06 g bromophenol blue and 0.06 g xylene cyanol. m CRITICAL Store at 4 1C in the dark or at C020 1C for up to 6 months. Formamide loading dye for LI-COR gels Mix 30 g AG50 1-X8 mixed-bed resin, 480 ml formamide, 20 ml 0.5 M EDTA (pH 7.5). Stir for 20 min. Add 40 mg bromophenol blue, mix and filter.mCRITICAL Store at 4 1C in the dark or at C020 1C for up to 6 months. 63 Loading dye for agarose gel Dissolve 0.025 g xylene cyanol in 5 ml of water. Add 3 ml glycerol and adjust to a final volume of 10 ml with water. m CRITICAL Store at 4 1C in the dark or at C020 1C for up to 6 months. Radiolabeled selective EcoRI primers see Box 1 and Ta b l e 1. EQUIPMENT SETUP Casting gels (see Box 2). PROCEDURE Checking DNA quality C15 TIMING Approximately 1 h 1| Check the integrity of the DNA. Run a small aliquot of DNA on a 1% (wt/vol) agarose gel in 1C2 TAE running buffer at 100 V for 10?15 min. Visualize using either EtBr or SYBR Safe DNA stain. Intact total DNA will have sharp high molecular weight band. Degraded DNA will appear as a low molecular weight smear. ? TROUBLESHOOTING p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h BOX 1 | RADIOLABELING PRIMERS FOR SELECTIVE AFLP AMPLIFICATION To allow TDF detection using a conventional autoradiography platform (Step 9A), one of the selective primers for selective AFLP amplification (Step 8) is radiolabeled by phosphorylating the 5�-end of the primer with [g- 33 P]ATP and polynucleotide kinase, as follows: 1. Incubate the labeling mix detailed in Table 1 for 45 min at 37 1C. 2. Stop the reaction by holding the temperature at 80 1C for 10 min. The labeled primer can be stored for up to 1 month at C020 1C. 1392 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL Template preparation C15 TIMING Approximately 4 h 2| Incubate 10 ml genomic DNA (approximately 0.1?0.5 mg) with 30 ml of the restriction digestion mix shown in Table 2 for 1 h at 37 1Candmixgently: ! CAUTION Prolonged incubation with the restriction enzyme EcoRI (e.g., overnight) is not recommended because of its possi- ble ?star? activity, giving reduced cleavage specificity and, ultimately, aberrant AFLP fingerprints. 3| Add 10 ml ligation mix as detailed in Table 3 and continue the incubation for another 3 h at 37 1C: Note: Do not inactive the restriction enzymes before the ligation. 4| After ligation, dilute the reaction mixture to 200 mlwithT 10 E 0.1 buffer. This will serve now as template for the pre-amplifi- cation reaction. ?PAUSE POINT If necessary, the template can be stored for up to 1 year at C020 1C. Pre-amplification C15 TIMING Approximately 3 h 5| Add 45 ml of the pre-amplification mix detailed in Table 4 (for an EcoRI/MseI primer pair) to 5 ml of the AFLP template prepared in Step 4. 6| Use the following PCR program: Cycle number Denature Anneal Extend 1?25 94 1C, 30 s 56 1C, 1 min 72 1C, 1 min ?PAUSE POINT The reaction mixture can be stored for up to 1 year at C020 1C. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h TABLE 1 | Primer radiolabeling mix. Volume to add (ll) for number of samples (X) Compound X � 50 X � 100 EcoRI+N 1?3 a primer (50 ng ml C01 )5 1 [g- 33 P]ATP (370 MBq ml C01 T4 polynucleotide kinase (10 U ml C01 )1 2 10C2 T4 buffer 2.5 5 Water 11.5 23 Final volume 25 50 a N represents a number of selective nucleotides that may be added; see section ??Experimental design?? in the INTRODUCTION for details of primer design. BOX 2 | CASTING GELS C15 TIMING APPROXIMATELY 1 H 30 MIN The AFLP reaction products are analyzed on 4.5% denaturing polyacrylamide gels or 6% Long Ranger gels. If conventional gel electrophoresis is to be used to detect radiolabeled products, cast 4.5% denaturing polyacrylamide gel according to option A. If a LI-COR automated DNA sequencer is to be used with infrared dye (IRD) technology for detection, cast a 6% Long Ranger gel by following option B. (A) Casting 4.5% denaturing polyacrylamide gels to detect radiolabeled products (i) Cast the gel according to the manufacturer?s instructions at least 2 h before use to ensure the proper polymerization of the gel. We prefer the SequiGenGT gel apparatus (38 C2 50 C2 0.04 cm 3 ), but there is no reason other sequencing gel systems should not work equally well. The back plate of the gels, the so-called integrated plate chamber, is treated with 2 ml of Repel-Silane. In case the gels need to be fixed, the front plate is treated with 10 ml of Bind-Silane solution. The Bind-Silane treatments cause the gels to stick to the front plate upon disassembly of the gel cassette after electrophoresis. (ii) The SequiGenGT sequence gels require approximately 100 ml of 1C2 4.5% denaturing polyacrylamide gel solution to which 500 mlof10% ammonium persulfate (APS) and 100 mlofTEMEDisadded. m CRITICAL Make sure to add the APS solution and TEMED immediately before pouring the gel, because these polymerize the gel. ! CAUTION Acrylamide and bis-acrylamide are highly neurotoxic. When handling these chemicals, wear gloves and use a pipetting aid. (B) Casting 6% Long Ranger gels for LI-COR analysis (i) Cast the 25-cm long sequencing gel (0.25-mm spacer thickness) according to the manufacturer?s instructions at least 2 h before use. This ensures sufficient time for gel polymerization. (ii) The Long Ranger gel requires 25 ml of 6% Long Ranger gel solution to which 166.5 ml of APS 10% and 16.5 mlTEMEDisadded. m CRITICAL Make sure to add the APS solution and TEMED immediately before pouring the gel, because these polymerize the gel. ! CAUTION Acrylamide and bis-acrylamide are highly neurotoxic. When handling these chemicals, wear gloves and use a pipetting aid. NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1393 PROTOCOL 7| Run 5 ml of the pre-amplification reaction product on a 1% agarose gel in 1C2 TAE running buffer at 100 V for 10?15 min. Use SmartLadder SF as molecular weight marker ranging from 100 to 1,000 bp. Use EtBr or SYBR Safe DNA stain to visualize the pre-amplification products. Substantial smearing in the range of 50?500 bp indicates a successful pre-amplification PCR. ? TROUBLESHOOTING 8| Dilute the pre-amplification reaction product obtained in Step 6 20-fold with T 10 E 0.1 buffer. These diluted reaction products serve as templates for the final selective amplification reactions using primers with two or three selective bases in one or both primers. Selective amplification C15 TIMING Approximately 3 h 9| Selective amplification can be accomplished using either radiolabeled primers (option A, to allow subsequent detection using the conventional autoradiography platform) or IRD-labeled primers (option B, to allow subsequent detection using an automated LI-COR platform). (A) Selective amplification using radiolabeled primers (i) Add 15 ml of the selective amplification mix shown in Table 5 to 5 ml of diluted pre-amplification reaction mixture from Step 8. (ii) Use the following PCR program: Cycle number Denature Anneal Extend 1?13 94 1C, 30 s 65 1C, 30 s (reduced each cycle by 0.7 1C) 72 1C, 1 min 14?36 94 1C, 30 s 56 1C, 30 s 72 1C, 1 min (B) Selective amplification using IRD-labeled primers (i) Add 15 ml of the selective amplification mix shown in Table 6 to 5 ml of diluted pre-amplification reaction mixture from Step 8. (ii) With IRD-labeled primers, the selective PCR profile is modified slightly to increase the relative intensity of larger fragments 28 , and the following PCR program is used: Cycle number Denature Anneal Extend 1?13 94 1C, 10 s 65 1C, 30 s (reduced each cycle by 0.7 1C) 72 1C, 1 min 14?36 94 1C, 10 s 56 1C, 30 s 72 1C, 1 min (extended 1 s per cycle) 37 72 1C, 2 min Electrophoresis and detection 10| Amplification products can be detected using either a conventional autoradiography platform (option A) or an automated LI-COR platform (option B). p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h TABLE 2 | Restriction digestion mix for template preparation. Compound Volume to add (ll) for number of samples (X) X � 10 X � 50 X � 70 X � 100 EcoRI (20 U ml C01 ) 2.5 12.5 17.5 25 MseI(10Uml C01 ) 5 25 35 50 10C2 RL buffer 40 200 280 400 Water 252.5 1,262.5 1,767.5 2,525 Final volume 300 1,500 2,100 3,000 TABLE 3 | Ligation mix for template preparation. Compound Volume to add (ll) for number of samples (X) X � 10 X � 50 X � 70 X � 100 EcoRI adapter (5 pmol ml C01 )10 50 70 1 MseI adapter (50 pmol ml C01 10C2 RL buffer 10 50 70 100 T4 DNA ligase (1 U ml C01 )10 50 1 ATP (10 mM) 10 50 70 100 Water 50 250 350 500 Final volume 100 500 700 1,000 1394 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL (A) Product detection using conventional autoradiography platform C15 TIMING Approximately 2 h 30 min (electro- phoresis) + 12?72 h (detection) (i) Mix the selective amplification reaction products from Step 9A(ii) with an equal volume (20 ml) of formamide loading dye. Mix carefully and store overnight at C020 1C. ? PAUSE POINT The mixture can be stored for up to 2 weeks at C020 1C. (ii) Cast a 4.5% denaturing polyacrylamide gel (see Box 2). ? TROUBLESHOOTING (iii) Fill the upper buffer tank with 1C2 Maxam buffer. For the lower buffer tank dissolve 8.8 g NaAc in 400 ml 1C2 Maxam buffer. This warrants no running off of smaller AFLP fragments. (iv) Pre-run the gel for 15 min at 100 W to warm up the gel to approximately 50?55 1C. This temperature is maintained through electrophoresis. (v) Denature the samples at 90 1C for 3 min and cool on ice. (vi) Rinse the surface of the gel well with 1C2 TBE using a syringe and needle. Push sharkstooth combs carefully approximately 0.5 mm into the gel surface to create the gel slots. (vii) Load 1.6?2.0 ml sample depending on the comb used (48, 64 or 96 wells). Load the molecular weight marker preferably in the first lane. If two or more PCs are run in parallel on one gel, load the molecular weight marker preferably in the lanes preceding the first sample lanes. (viii) Perform electrophoresis at constant power, 100 W for approximately 150 min. A constant temperature of 50?55 1C throughout electrophoresis is favorable. (ix) After electrophoresis, disassemble the gel cassette and either place the gel on blotting paper, covered with plastic (Saran), and dry for 1 h on a vacuum dryer at 75 1C or fix on the glass plate by soaking in 10% HAc for 30 min, rinsing with water for 10 min and drying at elevated temperatures in a fume hood. ? TROUBLESHOOTING (x) Autoradiograph the gel by exposing to a standard X-ray film for 2?3 d. Exposure times are reduced to 12 h when using phosphorimaging technology. (xi) Develop autoradiograph or visualize the fingerprint patterns using phosphorimager technology. ? TROUBLESHOOTING (B) Product detection using an automated LI-COR platform C15 TIMING Approximately 2 h 30 min (i) Mix 6 ml of the selective amplification reaction products from Step 9B(ii) with 3 ml of formamide loading dye. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h TABLE 4 | Pre?amplification mix for the pre?amplification step. Compound Volume to add (ll) for number of samples (X) X � 10 X � 50 X � 70 X � 100 EcoRI + N a primer (50 ng ml C01 ) 15 75 105 150 MseI+N a primer (50 ng ml C01 ) 15 75 105 150 AmpliTaq (5 U ml C01 ) 2 10 14 20 10C2 PCR buffer 50 250 350 500 MgCl 2 (25 mM) 50 250 350 500 dNTP mix (5 mM) 20 100 140 200 Water 298 1,490 2,086 2,980 Final volume 450 2,250 3,150 4,500 a N represents a number of selective nucleotides, either 1 or 2, that may be added; see section ??Experimental design?? in the INTRODUCTION for details ofprimerdesign. TABLE 5 | Selective amplification mix for the selective amplification step using radiolabeled primers. Compound Volume to add (ll) for number of samples (X) X � 10 X � 50 X � 70 X � 100 [g- 33 P]-labeled a EcoRI+N b primer (10 ng ml C01 ) 5 25 35 50 MseI+N a -primer (5 ng ml C01 )60304206 AmpliTaq (5 U ml C01 ) 1.2 6 8.4 12 10C2 PCR buffer 20 100 140 200 MgCl 2 (25 mM) 20 100 140 200 dNTP mix (5 mM) 8 40 56 80 Water 35.8 179 250.6 358 Final volume 150 750 1,050 1,500 a Procedure for radiolabeling primers can be found in Box 1 and Table 1. b N represents a number of selective nucleotides, either 1,2 or 3 that may be added; see section ??Experimental design?? in the INTRODUCTION for details of primer design. NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1395 PROTOCOL (ii) Cast a 6% Long Ranger gel (see Box 2). ? TROUBLESHOOTING (iii) Fill buffer tanks with running buffer 1C2 TBE. (iv) Pre-run the gel for 15 min at 45 W, 1,500 V, 40 mA and 45 1Ctowarmupthegel. (v) Denature the samples at 95 1C for 3 min and cool on ice. (vi) Rinse the surface of the gel well with 1C2 TBE using a syringe and needle. Push sharkstooth combs carefully approximately 0.5 mm into the gel surface to create the gel slots. (vii) Load 0.5?1.0 ml of each sample. Loaded volume depends on the comb used (48, 64 or 96 wells). Load the molecular weight marker preferably in the first lane. If two or more PCs are run in parallel on one gel, load the molecular weight mar- ker preferably in the lanes preceding the first sample lanes. (viii) Perform electrophoresis at 45 W, 1,500 V, 40 mA and 45 1C for 150 min (run time) and scan speed ?moderate?. Digital images are similar in appearance to the autoradiographs or phosphorimages produced with the conventional radiolabeling/ standard sequencing gel protocol. ? TROUBLESHOOTING C15 TIMING Step 1, checking DNA quality: approximately 1 h Steps 2?4, template preparation: approximately 4 h Steps 5?8, pre-amplification: approximately 3 h Step 9, selective amplification: approximately 3 h Step 10A, product detection: approximately 2 h 30 min electrophoresis + 12?72 h detection; Step 10B: approximately 2 h 30 min ? TROUBLESHOOTING Troubleshooting advice can be found in Table 7. p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h TABLE 6 | Selective amplification mix for the selective amplification step using infrared dye 700 (IRD 700)?labeled primers. Compound Volume to add (ll) for number of samples (X) X � 10 X � 50 X � 70 X � 100 IRD700-labeled EcoRI+N a primer (1 pmol ml C01 ) 8 40 56 80 MseI+N a primer (10 ng ml C01 )301502103 AmpliTaq (5 U ml C01 ) 2 10 14 20 10C2 PCR buffer 20 100 140 200 MgCl 2 (25 mM) 12 60 84 120 dNTP mix (5 mM) 8 40 56 80 Water 70 350 490 700 Final volume 150 750 1,050 1,500 a N represents a number of selective nucleotides, either 1, 2 or 3 that may be added; see section ??Experimental design?? in the INTRODUCTION for details ofprimerdesign. TABLE 7 | Troubleshooting table. Step Problem Possible cause Solution 1 DNA is not of sufficient quality DNA extraction was not performed properly Retry DNA extraction to get higher-quality DNA 7 No pre-amplification product No template Check concentration of adapters and/or starting amount of DNA and generate new template Amplification failed Check amplification mix, concentration of primers and repeat the amplification 10A(ii) and 10B(ii) Air bubbles in the polymerized gel Glass plates were not sufficiently clean(ed) Clean glass plates thoroughly with soap. When re-using glass plates, gel remnants might be present. Remove gel remnants from glass plate when they are still moist Polyacrylamide gel does not polymerize well Ammonium persulfate (APS) lost activity TEMED lost its catalytic activity Always use freshly made APS solution Use TEMED within manufacturer?s recom- mended expiration date. Store TEMED bottle closed and in dark 1396 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL ANTICIPATED RESULTS Figures 3a and 4 provide typical examples of AFLP gel images of segregating mapping populations and their parental lines (lanes 1 and 2) fingerprinted using this protocol and visualized using phosphorimaging technology (Fig. 3a) and the automated LI-COR platform (Fig. 4). Typically, 50?100 AFLP fragments are amplified and visualized in one single lane, and fragment size ranges from 50 to 500 bp, although a greater size range can generally be visualized and resolved with LI-COR automated sequencers (Fig. 4) than with the conventional sequencing gel electrophoresis (Fig. 3a). Digital images from the LI-COR sequencer are similar in appearance to the autoradiographs or phosphorimages with the conventional radiolabeling/standard sequencing gel electrophoresis. In contrast to conventional sequencing gels, all the fragments loaded on automated sequencers travel the same distance from the well before passing through the scan window. The bands representing the smallest fragments are therefore sharper and closer together than those representing the largest fragments on LI-COR gel images. The DNA fragments on conventional sequencing gels all spend the same amount of time in the gel but do not travel the same distance. The bands representing the largest fragments are therefore consequently sharper. Only AFLP fragments for which the parental lines are polymorphic segregate in the mapping population. Because genomic polymorphisms manifest themselves predominantly as single-base mutations that affect either the restriction site or the selec- tive nucleotides immediately adjacent to them, such polymorphisms result in a dominant PCR phenotype: the presence of a mutation causes the loss of an AFLP fragment from a fingerprint. Therefore, most AFLP markers are ?mono-allelic? markers because only one allele is actually visualized as a band in the fingerprint pattern. In contrast, an insertion/deletion polymorphism located in the internal sequence of an AFLP fragment results in a co-dominant PCR phenotype: the presence of an insertion/ deletion causes a size difference between the two AFLP marker alleles, both of which are visualized as a band in the fingerprint pattern, showing a complementary segregation pattern. Such bi-allelic AFLP markers are identified at a much lower frequency than mono-allelic AFLP markers. Examples of segregating mono- and bi-allelic markers are shown in Figure 4. Figure 3a represents a gel image of a tomato F 2 population, where the expected proportion of individuals heterozygous at a locus is 50%. The difference between samples homozygous (2n) or heterozygous (1n) for an AFLP marker can often be clearly distinguished from the band intensities (also by eye) and reflects PCR product concentrations (100 and 50%, respectively). This feature allows co-dominant scoring of AFLP markers based on relative fragment intensities with the aid of specific image analysis software AFLP-QuantarPro. Figure 4 represents a gel image of an AFLP analysis of 56 Arabidopsis Recombinant Inbred Line (RIL) offspring and their parental lines (in lanes 1 and 2). Given that RILs are panels of genetically mosaic but homozygous strains generated by crossing parental strains and inbreeding the progeny, AFLP markers segregate as homozygous present or absent in the RIL progeny. The scoring process of AFLP gels thus results in datasets consisting of dominantly scored markers, co-dominantly scored markers (Fig. 3b) or combinations thereof, depending on the population type involved and specifications of the scoring software used. These AFLP genotyping datasets typically serve as the starting point for further analysis in the context of the specific applica- tions for which the AFLP fingerprints are generated (often involving dedicated software packages). For instance, in the case of genetic linkage mapping, AFLP marker datasets are used to group and order the AFLP markers in linkage groups and estimate genetic distances between them using genetic linkage mapping software. Such linkage maps are useful tools to identify genes affecting (complex) traits for breeding purposes. Another widely used application in plants is marker-assisted back-crossing. In this case, AFLP marker genotypes are used to estimate the fraction of donor genome and the number of donor fragments that are introgressed into the recurrent parent line of each back-cross progeny, such that those progeny with the lowest number of p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h 10A(ix) Gels stick to IPC upon disas- sembly of the gel cassette Avoid any contact between Bind-Silane and IPC. Treat the IPC thoroughly with Repel- Silane 10B(viii) Poor resolution of bands in LI- COR-generated fingerprint Bromophenol blue quenches the infrared dye signal Lower concentration of bromophenol blue in loading dye or order new bromophenol blue 10A(xi) and 10B(viii) No fingerprint No template Repeat dilution of pre-amplification reaction product obtained in Step 6 and repeat amplification Labeling failed Repeat labeling Amplification failed Check amplification mix, concentration of primers and repeat the amplification TABLE 7 | Troubleshooting table (continued). Step Problem Possible cause Solution NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1397 PROTOCOL donor segments can be selected for the next generation of back-cross breeding. By repeating this process, the number of gene- rations required for introgression of only a single donor fragment into elite cultivars can be reduced significantly. Yet another application of AFLP is the saturation of genomic regions with markers using pooled DNA samples obtained from (plant or animal) samples differing with respect to a phenotype of interest. By subjecting these pooled samples to AFLP fingerprinting, AFLP markers are discovered that are expected to be located near genetic loci that control the phenotype. This combination of AFLP with bulked segregant analysis 29 is a powerful approach for marker development, especially in species for which little sequence information is known. Finally, AFLP analysis is also widely used to estimate genetic relatedness of samples within a species across a wide range of taxa (plant, animal, micro-organism). Most of these applications are attractive because of the combination of the high multiplexing level of AFLP (and therefore low cost per data-point) and the ability to apply the technique without prior sequence knowledge according to a fixed protocol. ACKNOWLEDGMENTS We are grateful to H. Van den Daele and I. Vercauteren for their help with the manuscript. The AFLP technology is covered by patents and patent applications owned by Keygene N.V. AFLP and AFLP-QuantarPro are registered trademarks of Keygene N.V. All other product names, brand names or company names are used for identification purposes only and may be (registered) trademarks of their respective owners. COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the HTML version of this article for details). Published online at http://www.natureprotocols.com Rights and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Vos, P. et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407?4414 (1995). 2. Heun, M. et al. Site of einkorn wheat domestication identified by DNA fingerprinting. Science 278, 1312?1314 (1997). 3. Janssen, P. etal. 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Identification of AFLP markers associated with round heart syndrome in turkeys. Int. J. Poultry Sci. 4, 133?137 (2005). 27. de Gruijter, J.M., Gasser, R.B., Polderman, A.M., Asigri, V. & Dijkshoorn, L. High resolution DNA fingerprinting by AFLP to study the genetic variation among Oesophagostomum bifurcum (Nematoda) from human and non-human primates from Ghana. Parasitology 130, 229?237 (2005). 28. Myburg, A.A., Remington, D.L., O?Malley, D.M., Sederoff, R.R. & Whetten, R.W. High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer. Biotechniques 30, 348?357 (2001). 29. Michelmore, R.W., Paran, I. & Kesseli, R.V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88, 9828?9832 (1991). p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 � naturepr otocol s / m o c . e r u t a n . w w w / / : p t t h 1398 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL CORRIGENDUM Corrigendum: AFLP technology for DNA fingerprinting Marnik Vuylsteke, Johan D Peleman & Michiel JT van Eijk Nat. Protoc. 2, 1387?1398 (2007); doi:10.1038/nprot.2007.175; published online 31 May 2007; corrected online 14 August 2008. In the version of this article initially published, the three genotype classes in Figure 3b were incorrectly described in the legend. ?A: homozygous as the ?rst parent; H: heterozygous, B: homozygous as the second parent? should have read ?A: homozygous absent; H: heterozygous; B: homozygous present?. This error has been corrected in the HTML and PDF versions of the article. NATURE PROTOCOLS � 200 8 Nature Pub lishing Gr oup http://www .nature .com/n atureprotocols "
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