AFLP Microbial Fingerprinting Protocol (PN 402977F)
Contents
1. GeneScan 500 Size Standard The GeneScan 500 standard is made of double stranded DNA fragments but only one of the strands is labeled with an ABI PRISM dye Consequently under denaturing conditions even if the two strands migrate at different rates only the one labeled strand is detected Because of this you can avoid split peaks which result when two strands move through a denaturing gel at different rates Under denaturing conditions you can achieve a linear range of separation for fragment sizes of up to 500 bases Figure 8 on page 26 25 EN 65500 ROH 50 90 120 150 180 210 240 270 300 330 380 390 420 450 490 h n n n i i i h i n i TM 2R 03 HYPFINE19 M z25 v i188 p Figure 8 Electropherogram of GeneScan 500 run under denaturing conditions Using the Standard Sizing Curve The Standard Sizing Curve is a measure of how well the standard definition matches the GeneScan size standard and whether or not it is linear To align the data by size GeneScan calculates a best fit least squares curve for all samples This is a third order curve when you use the Third Order Least Squares size calling method For all other size calling methods it is a second order curve Displaying the Standard Sizing Curve Step Action 1 Select a sample or multiple samples in the Analysis Control or Results Control window To select sev2. Unsuccessful amplification faint or no peaks Incomplete restriction ligation Repeat restriction ligation with fresh enzymes and buffer Use an agarose gel to check PCR inhibitors may exist in the DNA sample Try different extraction procedures Use an agarose gel to check Insufficient or excess template DNA Use recommended amount of template DNA Use an agarose gel to check If DNA is stored in water check water purity Insufficient enzyme activity Use the recommended amount of restriction digestion enzyme ligase and AmpliTaq DNA Polymerase TE 4 buffer not properly made or contains too much EDTA Add appropriate amount of MgCl solution to amplification reaction Remake the TE Incorrect thermal cycling parameters Check protocol for correct thermal cycling parameters High salt concentrations of K Na or Mg Use correct amount of DNA and buffer High salt and glycerol can inactivate restriction ligation enzymes Incorrect pH Use correct amount of DNA and buffer Tubes loose in the thermal cycler Push reaction tubes firmly into contact with block before first cycle Wrong style tube Use Applied Biosystems GeneAmp Thin Walled Reaction Tubes and DNA Thermal Cycler 480 or MicroAmp Reaction Tubes with Cap forthe GeneAmp PCR System 9600 or System 2400 Primer concentration too low Use recommended primer concentration Ligase ina
3. tpe tees 19 Selective Amplification cece 19 Evaluating Results ea cree mE ode Ae a etta 21 21 Preparing the Loading Buffer for the ABI 373 and ABI PRISM 377 21 Loading and Electrophoresis on the ABI 373 and ABI PRISM 377 22 Preparing the Loading Buffer for the ABI PRISM 310 23 Loading and Electrophoresis on the ABI PRISM 310 23 Using GeneScan to Analyze Results 24 Evaluating ABI 373 DNA Sequencer Results 29 Evaluating ABI PRISM 377 DNA Sequencer Results 30 Evaluating ABI PRISM 310 Genetic Analyzer Results 31 Appendix A Troubleshooting eese 32 Appendix B References eee eee 35 Appendix C Related Reagents Consumables and Accessories 38 Appendix D Technical 58000016 42 Contacting Technical Support esee 42 To Contact Technical Support by E Mail 42 Hours for Telephone Technical Support 04 42 To Contact Technical Support by Telephone or Fax 43 To Reach Technical Support Through the Internet 46 To Obtain Documents on 2 47 MU Sh Ae Eg Bl netted 47 Introduction What is AFLP Advantages of AFLP Applicati
4. 402956 500 uL 402032 500 uL EcoRI AG JOE 402268 250 uL EcoRI ACG JOE 402044 250 uL 402957 500 uL 402036 500 uL EcoRI AT NED 402955 500 uL EcoRI ACT FAM 402045 250 uL 402037 500 uL EcoRI AT TAMRA 402270 250 uL EcoRI AGC TAMRA 402041 250 uL 402033 500 uL EcoRI TA JOE 402267 250 uL EcoRI AGG JOE 402043 250 uL 402035 500 uL 38 Table 9 AFLP Msel selective amplification primers Primer Part Number Primer Part Number Msel 0 402958 500 uL Msel CAA 402021 250 uL 402029 500 uL Msel A 402959 500 uL Msel CAC 402020 250 uL 402028 500 uL Msel C 402961 500 uL Msel CAG 402019 250 uL 402027 500 uL Msel G 402962 500 uL Msel CAT 402018 250 uL 402026 500 uL Msel T 402960 500 uL Msel CTA 402017 250 uL 402025 500 uL Msel CA 402963 500 uL Msel CTC 402016 250 uL 402024 500 uL Msel CC 402965 500 uL Msel CTG 402015 250 uL 402023 500 uL Msel CG 402966 500 uL Msel CTT 402014 250 uL 402022 500 uL Msel CT 402964 500 uL Table 10 AFLP Plant Mapping Kit Modules Regular Plant Small Plant Genomes Genomes Module 500 6000 Mb 50 500 Mb Ligation and P N 402004 P N 402273 Preselective Amplification Selective Amplification P N 402006 P N 402272 Start Up Table 11 Related consumables and accessories Name Description Vendor AFLP Protocol Reagents and Equipment AFLP Microbial Adaptor Core
5. Sequences Consists of AFLP EcoRI and Msel adaptor pairs and core sequences Applied Biosystems P N 402943 E coli W3110 DNA Reference DNA Applied Biosystems P N 402990 T4 DNA ligase New England Biolabs T4 DNA ligase buffer New England Biolabs EcoRI restriction enzymes Use higher concentration formulations of vendor supplied enzymes New England Biolabs Msel restriction enzymes Use higher concentration formulations of vendor supplied enzymes New England Biolabs Bovine serum albumin BSA Nuclease free Dilute 10 mg mL solution supplied by vendor to 1 0 mg mL New England Biolabs 6 Pre mixed Gel matrices for the ABI Amresco polyacrylamide with 373 DNA Sequencer 7 5Murea in TBE buffer LongRanger gel AT Biochem formulations JT Baker solutions Used for the ABI PRISM 377 DNA Sequencer at P N 4730 02 for 250 mL 5 or 6 in TBE buffer Performance Polymer solution used Applied Biosystems Optimized Polymer 4 with the ABI PRISM310 P N 402838 POP 4 10X TBE buffer stock Gibco Deionized formamide Applied Biosystems P N 400596 Gel loading pipette tips 0 17 mm flat for the ABI PRISM 377 Rainin P N GT 1514 Table 11 Related consumables and accessories continued Name Description Vendor Standards GeneScan 500 ROX Size standard Internal lane size standard labeled on a single strand with ROX NHS ester dye
6. G Msel A EcoRI C Msel C EcoRI A Msel T EcoRI G Msel A EcoRI T Msel C EcoRI 0 Msel C EcoRI A Msel C EcoRI G Msel A EcoRI T Msel C EcoRI A Msel CA EcoRI AC Msel C EcoRI A Msel G EcoRI AC Msel C EcoRI A Msel T EcoRI G Msel A EcoRI C Msel A EcoRI AC Msel C EcoRI A Msel CA EcoRI AC Msel C EcoRI AC Msel C EcoRI 0 Msel C EcoRI 0 Msel A EcoRI 0 Msel G EcoRI 0 Msel A a Producing 25 130 bands evenly dispersed from 50 500 bases with intensities of 100 2000 relative fluorescent units b Too few or too many bands or uneven size distribution Fluorescent Dye labeling and Marker Detection Note The list in Table 2 on page 8 is not exhaustive Refer to the publications listed in Appendix B on page 35 for in depth discussion of primer choices Applied Biosystems has adapted the AFLP technique for use with its ABI PRISM fluorescent dye labeling and detection technology PCR products are dye labeled during amplification using a 5 dye labeled primer For high throughput you can co load up to three different reactions labeled with different colored dyes in a single lane on the ABI 373 or ABI PRISM 377 DNA Sequencer or in a single injection on the ABI PRISM 310 Genetic Analyzer Load an internal lane size standard with a fourth color in every lane to size all amplification fragments accurately You can automate the scoring of the large numbers of markers that are typically generated by analyzing your results w
7. L Aucken H Gerner Smidt P Janssen P Kaufmann M E Garaizar J Ursing J and Pitt T L 1996 Comparison of outbreak and nonoutbreak Acinetobacter baumanii strains by genotypic and phenotypic methods Journal of Clinical Microbiology 34 1519 1525 Folkertsma R T Rouppe van der Voort J N A de Groot K E van Zandvoort P M Schots A Gommers F J Helder J and Bakker J 1996 Gene pool similarities of potato cyst nematode populations assessed by AFLP analysis Molecular Plant Microbe Interactions 9 47 54 Heyndrickx M Vandemeulebroecke K Hoste B Janssen P Kersters K Vos P Logan N A Ali N and Berkeley R C W 1996 Reclassification of Paenibacillus formerly Bacillus pulvifaciens a later subjective synonym of Paenibacillus formerly Bacillus larvae as a subspecies of P larvae with emended descriptions of P larvae as 35 P larvae subsp larvae and P larvae subsp pulvifaciens International Journal of Systematic Bacteriology 46 270 279 Huys G Coopman R Janssen and Kersters K 1996 High resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting International Journal of Systematic Bacteriology 46 572 580 Janssen P Coopman R Huys G Swings J Bleeker M Vos P Zabeau M and Kersters K 1996 Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy Microbiol
8. PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Telephone Fax Region Dial Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Telephone Fax Region Dial Dial Eastern Asia China Oceania Australia Scoresby 61 3 9730 8600 61 3 9730 8799 Victoria China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 75 0 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 2 61 222 168 D
9. primers The JOE labeled EcoRI fragments are displayed as peaks in the electropherogram Em EE 60 30 120 n 180 210 240 270 300 330 360 3390 420 450 480 510 1 jl Aud l A AA Figure 11 Electropherogram of AFLP samples run on an ABI 373 continued on next page 29 Evaluating ABI PRISM 377 DNA Sequencer Results A representative electropherogram of fluorescent dye labeled AFLP products run on an ABI PRISM 377 DNA Sequencer and analyzed using GeneScan analysis software is shown in Figure 12 The analyzed products are DNA fragments amplified with Msel and FAM dye labeled EcoRI selective amplification primers The FAM labeled EcoRI fragments are displayed as peaks in the electropherogram a 30 60 30 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 1 OMA vi T li M M WN a eed Red oe 26B ENSF m B hoan vises o Figure 12 Electropherogram of AFLP sample run on an ABI PRISM 377 Figure 13 on page 31 shows an expanded electropherogram of select peaks from the same AFLP samples shown in Figure 12 Tabular data in Figure 13 shows the sizes of sample fragments in mobility units All sample fragments were sized using the GeneScan 500 ROX size standard Electropherogram
10. species or strain level high resolution genotyping for taxonomic applications detection of DNA polymorphisms in genome evolution studies determining the relatedness of pathogenic organisms in epidemiological studies mapping of cloned fragments in bacterial and yeast artificial chromosomes BACs and YACs An example of AFLP fingerprints is shown in Figure 1 The first 24 lanes show six samples each of four different Escherichia coli strains each of the six samples represents a different growth phase of the organism The final 11 lanes show different growth phases of a single strain of Legionella pneumophila Note that the E coli fingerprints are similar to each other and different from the Legionella fingerprint Within a strain all of the bands are reproducible E coli strains Legionella strain Figure 1 AFLP fingerprints of four E coli strains and one Legionella strain Large population studies provide data for the linkage of a band with a given phenotype such as pathogenicity For examples of other applications refer to the literature cited in Appendix B on page 35 The AFLP Technique Template Preparation and Adaptor Ligation Preselective Amplification The first step of the AFLP technique is to generate restriction fragments by using two restriction endonucleases EcoRI and Msel in the AFLP Microbial Fingerprinting Kit Double stranded adaptors supplied with each kit are ligated to the ends of the DNA fra
11. the display color of one or more of the samples so that the electropherograms are in different colors 3 Record the sizes of the polymorphic peaks and the samples that produced them Figure 10 shows the polymorphic peak patterns from a GeneScan analysis of two AFLP samples Polymorphic peaks are labeled with size and origin Results 1B 2B Stacked un u A BE Lane 1 Sample 1 BE Lane 2 Sample 2 a A S ceo Figure 10 Overlapping electropherograms for two AFLP samples You can import GeneScan results data into a Genotyper software template Used together GeneScan and Genotyper can automate segregation scoring of AFLP results For more information on how you can analyze polymorphic peaks using Genotyper see the Genotyper DNA Fragment Analysis Software User s Manual continued on next page Evaluating ABI If you run samples under the recommended electrophoresis conditions 373DNA and analyze them with GeneScan resulting electropherogram data Sequencer Results from the ABI 373 DNA Sequencer should look similar to data from samples run on the ABI PRISM 377 DNA Sequencer Figure 11 shows a representative electropherogram of fluorescent dye labeled AFLP products run on an ABI 373 DNA Sequencer and analyzed using GeneScan analysis software The analyzed products are DNA fragments modified with Msel and JOE dye labeled EcoRI selective amplification
12. van Enckevort E Segers B Peleman J Jacobsen E Helder J and Bakker J 1995 The inheritance and chromosomal location of AFLP markers in a non inbred potato offspring Molecular Breeding 1 397 410 Vos P Hogers R Bleeker M Reijans M van de Lee T Hornes M Fritjers A Pot J Peleman J Kuiper M and Zabeau M 1995 AFLP a new concept for DNA fingerprinting Nucleic Acids Research 23 4407 4414 Zabeau M and Vos P 1993 Selective Restriction Fragment Amplification A general method for DNA Fingerprinting European Patent Application EP 0534858 37 Appendix C Related Reagents Consumables and Accessories This appendix contains ordering information and descriptions of various reagents kits and consumables which you can use to perform procedures described in this protocol Table 8 AFLP EcoRI selective amplification primers Primer Part Number Primer Part Number EcoRI 0 FAM 402949 500 uL EcoRI TC FAM 402265 250 uL EcoRI A FAM 402950 500 uL EcoRI TG FAM 402264 250 uL EcoRI C NED 402952 500 uL EcoRI TT TAMRA 402266 250 uL EcoRI G JOE 402953 500 uL EcoRI AAC TAMRA 402039 250 uL 402031 500 uL EcoRI T JOE 402951 500 uL EcoRI AAG JOE 402042 250 uL 402034 500 uL EcoRI AA JOE 402271 250 uL EcoRI ACA FAM 402038 250 uL 402954 500 uL 402030 500 uL EcoRI AC FAM 402269 250 uL EcoRI ACC TAMRA 402040 250 uL
13. 7 Sterile 0 5 ml microcentrifuge tubes Thermal cycler Applied Biosystems Sterile 0 2 mL MicroAmp Thin Walled Reaction Tubes and caps GeneAmp PCR Instrument Systems 2400 and 9600 Sterile GeneAmp Thin Walled 0 5 mL Reaction Tubes DNA Thermal Cycler 480 13 Sample Preparation Before Starting an Before setting up an AFLP experiment determine whether or not your AFLP Experiment genomic DNA restricts properly with EcoRI and Msel Step Action 1 Digest 1 3 ug of DNA with the enzymes Msel and EcoRI separately then with both together according to the manufacturer s instructions Load the digestion products in one lane on a 1 5 mini agarose gel with size markers Stain with ethidium bromide WARNING Ethidium bromide is a powerful mutagen and is moderately toxic Wear gloves a lab coat and safety glasses when using this dye After use decontaminate ethidium bromide solutions before disposal View on a UV transilluminator For an example of what a successful digest looks like see Figure 6 on page 18 left half Preparing To prepare samples for the AFLP preselective and selective Samples for PCR amplification reactions you must Amplification 4 14 anneal the adaptor pairs prepare a restriction ligation enzyme master mix prepare the restriction ligation reactions dilute the restriction ligation reactions continued on next page Anneal Adaptor You
14. AFLP Microbial Fingerprinting Protocol Applied KS Biosystems Copyright 2007 2010 Applied Biosystems For Research Use Only Not for use in diagnostic procedures Notice to Purchaser Limited License Use of this product is covered by US patent claims and corresponding patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as the patented 5 Nuclease Process claims no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA The AFLP process is covered by patents or patent applications owned by Keygene N V This product is sold under license from Keygene N V This kit may be used only for research purposes The use of this kit for any activity other than research activities for the user s own benefit such other activitie
15. ARNING Chemical hazard Before handling any of the chemicals 12 Supplied listed below familiarize yourself with the Materials Safety Data Sheet MSDS Always follow safety precautions and wear proper protective equipment eye protection gloves lab coat Dispose of waste in accordance with all local state and national regulations Nuclease free distilled deionized water EcoRI restriction endonuclease 500 Units Msel restriction endonuclease 100 Units T4 DNA Ligase 100 Units 10X T4 DNA ligase buffer containing ATP see page 16 NaCl 0 5 M nuclease free molecular biology grade Bovine serum albumin BSA 1 0 mg mL nuclease free 1X TE 9 buffer 20 mM Tris HCl 0 1 mM EDTA pH 8 0 nuclease free 6 denaturing polyacrylamide gel for the ABI 373 DNA Sequencer 5 Long Ranger gel for the ABI PRISM 377 DNA Sequencer Performance Optimized Polymer 4 POP 4 Applied Biosystems P N 402838 for the ABI PRISM 310 Genetic Analyzer Deionized formamide Applied Biosystems P N 400596 GeneScan 500 ROX Size Standard Applied Biosystems P N 401734 DNA size markers e g Boehringer Mannheim set VI 9 9 9 9 9 9 9 Dye Primer Matrix Standard Kit Applied Biosystems P N 401114 NED Matrix Standard Applied Biosystems P N 402996 substitutes for TAMRA Equipment 9 9 9 9 9 Microcentrifuge Pipettors 2 uL 20 uL and 200 LL with sterile pipette tips Gel loading pipette tips 0 17 mm flat ABI PRISM 37
16. Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM9877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRISM9 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 43 Product or Product Area Telephone Dial Fax Dial Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855
17. R amplification with these primers a portion of the samples is analyzed on a Applied Biosystems DNA Sequencer Selective amplification with an EcoRI and an Msel primer amplifies primarily EcoRI Msel ended fragments The EcoRI EcoRI fragments do not amplify well The Msel Msel fragments are not visualized because they do not contain fluorescent dye labels Only the EcoRI containing strands are detected Figure 4 A Choose selective AFLP primers MEN o x AX one of nine different fluorescent dye labeled AFLP EcoRI selective amplification primers 0 X CX one of nine different AFLP Msel selective amplification primers B Run selective amplification m Primers l Thermal Core Mix Cycling m Figure 4 Selective amplification with fluorescent dye labeled primers Figure 5 on page 6 shows examples of AFLP fingerprint patterns that were prepared using different selective primers Note that the EcoRI selective primers with one nucleotide extensions EcoRI A EcoRI T and EcoRI G give simpler patterns than that obtained using the primer with no extra nucleotide EcoRI O Testing New Genomes 12B W3110 CAO 138 W3110 64 4 GM 110 3110 cats GM 156 ws110 cae Figure 5 AFLP fingerprints of E coli W3110 Reference DNA The Msel CA and fluorescent dye labeled EcoRI 0 EcoRI A EcoRI T and EcoRI G selective primers shown here top to bottom resp
18. Shipped in two tubes containing 200 uL of material each Sizes fragments between 35 and 500 bases Applied Biosystems P N 401734 Dye Primer Matrix Standard Kit Although FAM JOE and ROX fluoresce at different wavelengths there is some overlap in the emission spectra To correct for this overlap filter cross talk a mathematical matrix needs to be created and stored as a matrix file When data is analyzed the appropriate matrix is applied to the data to subtract out any emission overlap Applied Biosystems P N 401114 NED Matrix Standard See above NED substitutes for TAMRA as the yellow dye in the AFLP Microbial Fingerprinting Kit Applied Biosystems P N 402996 41 Appendix D Technical Support Contacting Technical Support To Contact Technical Support by E Mail Hours for You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems
19. age you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not already done so then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts within 24 to 48 hours To Obtain Free 24 hour access to Applied Biosystems technical documents Documents on including MSDSs is available by fax or e mail or by download from our Demand Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index nu
20. ation Check if buffer concentration matches protocol requirements Incorrect run temperature Check the Log for the record of the electrophoresis temperature Appendix B References Ausubel F M Brent R Kingstin R E Moore D D Seidman J G Smith J A and Struhl K eds 1987 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience John Wiley and Sons New York Bachem C W B van der Hoeven R S de Bruijn S M Vreugdenhil D Zabeau M and Visser R G F 1996 Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP analysis of gene expression during potato tuber development The Plant Journal 9 745 753 Ballvora A Hesselbach J Niewohner J Leister D Salamini F and Gebhardt C 1995 Marker enrichment and high resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1 Molecular and General Genetics 249 82 90 Bates S R E Knorr D A Weller J N and Ziegle J S 1996 Instrumentation for automated molecular marker acquisition and data analysis in Sobral B W S ed The Impact of Plant Molecular Genetics Birkha ser Boston MA pp 239 255 Becker J Vos P Kuiper M Salamini F and Heun M 1995 Combined mapping of RFLP and AFLP markers in barley Molecular and General Genetics 249 65 73 Dijkshoorn
21. com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers tsupport appliedbiosystems com LC MS Applied Biosystems MDS Sciex apisupport sciex com or api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com In the United States and Canada technical support is available at the Telephone following times Technical Support 42 Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981
22. ctive Check activity with control DNA 32 Table 7 Troubleshooting AFLP Procedures continued Observation Possible Causes Potential Solution Inconsistent results with control DNA Restriction incomplete Repeat the restriction ligation Incorrect PCR thermal profile program Choose correct temperature control parameters refer to the GeneAmp PCR System 9600 User s Manual GeneAmp PCR System 9600 misaligned lid Align 9600 lid white stripes after twisting the top portion clockwise For DNA Thermal Cycler 480 improper tube placement in block Refer to the DNA Thermal Cycler 480 User s Manual Pipetting errors Calibrate pipettes attach tips firmly and check technique Combined reagents not spun to bottom of tube Place all reagents in apex of tube Spin briefly after combining Combined reagents left at room temperature or on ice for extended periods of time Put tubes in block immediately after reagents are combined Extra peaks visible when sample is known to contain DNA from a single source Contamination with exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Incomplete restriction or ligation Extract the DNA again and repeat the restriction ligation Samples not denatured before loading in the autosampler Make sure the samples are heated at 95 C for three minutes prior to loa
23. d on next page 15 Prepare Restriction Ligation Reactions Dilute Restriction Ligation Reactions 16 The restriction ligation reactions prepare the template for adaptors and then ligate adaptor pairs to the prepared template DNA Step Action 1 Combine the following in a sterile 0 5 mL microcentrifuge tube 0 01 ug genomic DNA in 5 5 uL sterile distilled water or 1 0 uL of E coli W3110 reference DNA plus 4 5 uL sterile distilled water 1 0 uL 10X T4 DNA ligase buffer that includes ATP 1 0 uL 0 5M NaCI 0 5 uL 1 0 mg mL BSA dilute from 10 mg mL if necessary 1 0 uL Msel adaptor 1 0 uL EcoRI adaptor 9 9 9 9 9 1 0 uL Enzyme Master Mix Mix thoroughly and place in a microcentrifuge for ten seconds Incubate at room temperature overnight or for two hours at 37 C in a thermal cycler with a heated cover to prevent EcoRI star activity Note Be careful that the volume of enzyme added does not cause the amount of glycerol to be gt 5 which also leads to EcoRI activity Dilute the restriction ligation samples to give the appropriate concentration for subsequent PCR Step Action 1 Add 189 uL of TE buffer to each restriction ligation reaction 2 Mix thoroughly 3 Store the mixture at 2 6 C for up to one month or at 15 to 25 C for longer than a month Note The mixture can be used as the template for selective AFLP reactions
24. data and tabular data were generated using GeneScan Analysis software version 2 0 Evaluating ABI PRISM 310 Genetic Analyzer Results 3500 3000 2500 2000 1590 1000 o aN I r 188 NE FUN A Wu Ld M M ul aA y MEN 26B b nm v 272 d Due Sample Peak m 26B 49 265 50 265 51 26B 52 26B 53 26B 54 26B 55 266 56 26b 57 26B 58 IE E E E2 E E E EJ EJ E 26B 59 Minutes Figure 13 Expanded electropherogram and size data for AFLP sample An electropherogram of E coli W3110 Reference DNA run on an ABI PRISM 310 Genetic Analyzer is shown in Figure 14 The Msel CA and FAM labeled EcoRI A selective primers were used Note that the band pattern looks very similar to that shown in Figure 5 on page 6 There are slight differences because fragments size differently on the ABI PRISM 310 compared to the ABI 373 and ABI PRISM 377 S90 120 150 180 210 240 270 300 330 360 390 420 450 480 i Figure 14 ABI PRISM 310 electropherogram of E coli W3110 Reference DNA Wir 12B Samplee13 31 Appendix A Troubleshooting Table 7 Troubleshooting AFLP Procedures Observation Possible Causes Potential Solution
25. ding Renaturation of denatured samples Load sample immediately following denaturation or store on ice until ready Too much DNA in reaction so that insufficient adaptor present Use recommended amount of template DNA Too much DNA amplified and or loaded resulting in crossover between color channels Re run PCR using less DNA or load less sample during electrophoresis 33 Table 7 Troubleshooting AFLP Procedures continued Observation Possible Causes Potential Solution Signal continually gets weaker Outdated or mishandled reagents Check expiration dates on reagents Store and use according to manufacturers instructions Compare with fresh reagents Degraded primers Store unused primers at 15 to 25 C Do not expose fluorescent dye labeled primers to light for long periods of time Inconsistent sizing of known DNA sample Inadvertent change in analysis parameters Check settings for GeneScan analysis parameters Change in size calling method Use same size calling method Incorrect internal standard Use correct GeneScan size standard Change in electrophoresis temperature Check the Log for the record of the electrophoresis temperature Data was not automatically analyzed Sample Sheet not completed Complete Sample Sheet correctly Samples run faster than usual with decreased resolution Incorrect buffer concentr
26. ec 30 sec 2 min 94 C 64 C 72 C 1 20 sec 30 sec 2 min 94 C 63 C 72 C 1 20 sec 30 sec 2 min 94 C 62 C 72 C 1 20 sec 30 sec 2 min 94 C 61 C 72 C 1 20 sec 30 sec 2 min 94 C 60 C 72 C 1 20 sec 30 sec 2 min 94 C 59 C 72 C 1 20 sec 30 sec 2 min 94 C 58 C 72 C 1 20 sec 30 sec 2 min 94 C 57 C 72 C 1 20 sec 30 sec 2 min 94 C 56 C 72 C 20 20 sec 30 sec 2 min 60 C 1 30 min 4 C 1 forever Evaluating Results Overview Preparing the Loading Buffer for the ABI 373 and ABI PRISM 377 You can evaluate the results of the AFLP reactions by using GeneScan software to analyze data from samples loaded and run on the ABI 373 or ABI PRISM 377 DNA Sequencer or on the ABI PRISM 310 Genetic Analyzer The following instructions describe step by step procedures for loading samples and performing electrophoresis on these instruments Prepare a loading buffer mix of the following reagents in the proportions shown in sufficient quantity for each sample 1 25 uL deionized formamide 0 25 uL blue dextran 50 mM EDTA loading solution supplied with the size standard 05111 of GeneScan 500 ROX size standard WARNING Chemical hazard formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use chemical resistant gloves and safety glasses when handling Note You can store any r
27. ectively were used If the complexity of the AFLP pattern is still too high at the 2 2 level we recommend reamplifying the preselective amplification sample with the preselective primers from the AFLP Ligation and Preselective Amplification Modules of the AFLP Regular and Small Plant Genome Mapping Kits P N 402004 and 402273 respectively When testing novel genomes you must be sure that the DNA restriction digest with EcoRI and Msel generates enough fragments for comparison of samples There is a large variability in the number of restriction sites within microbial genomes No assurances of kit performance are made for organisms not listed Empirical guidelines suggest that if the G C content of the genome is 76596 Msel will not give a significant number of fragments Optimal results are obtained with Msel when the G C content is 5096 EcoRI also tends to produce more fragments in G C poor genomes In cases where an organism s G C content is unknown the effectiveness of the restriction enzymes must be determined empirically continued on next page Primer Selection For genomes that restrict well with the EcoRI and Msel restriction Guidelines endonuclease combination some general recommendations can be made in terms of the genome size and the selective nucleotides to choose for subsequent amplification Table 1 Table 1 Guide to choosing selective primers Nucleotide Application Addition EcoRI Primers Msel Prime
28. emaining loading buffer at 2 6 C for a week continued on next page 21 Electrophoresis on 22 Loading and the ABI 373 and ABI PRISM 377 For specific instructions about loading and running samples refer to the ABI 373 DNA Sequencing System User s Manual or the ABI PRISM 377 DNA Sequencer User s Manual Step Action 1 Add 2 0 uL of the loading buffer mix to a 0 2 mL MicroAmp Reaction Tube Use one tube for each sample 2 On the ABI 373 DNA On the ABI PRISM 377 DNA Sequencer Sequencer Add 1 0 uL of the selective Dilute the selective amplification amplification product to the product with two parts of TE tubes buffer Add 1 0 uL of the diluted product to the tubes 3 Heat tubes to 95 C for three minutes 4 Quick chill on ice On the ABI 373 DNA On the ABI PRISM 377 DNA Sequencer Sequencer Load the entire sample onto a Load 1 5 2 uL of the sample 6 denaturing polyacrylamide onto a 5 denaturing Long gel using 1X TBE running Ranger gel using 1X TBE buffer running buffer IMPORTANT Use Filter Set A with AFLP Microbial Fingerprinting Kit modules on the ABI 373 or ABI PRISM 377 DNA Sequencer Make the matrix with the Dye Primer Matrix Standards P N 401114 substituting the NED Matrix Standard P N 402996 for TAMRA Table 5 ABI 373 and ABI PRISM 377 Electrophoresis Parameters Well to read Limiting Instrument distance parameter Time ABI 373 24 cm 1680 volts 11 0 ho
29. enmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 2312 05 75 Poland Lithuania Latvia 48 22 866 40 10 48 22 866 40 20 and Estonia Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 45 To Reach Technical Support Through the Internet Telephone Fax Region Dial Dial United Kingdom 44 0 1925 825650 44 0 1925 282502 Warrington Cheshire All other countries not 44 0 1925 282481 44 0 1925 282509 listed Warrington UK Japan Japan Hacchobori 81 3 5566 6230 81 3 5566 6507 Chuo Ku Tokyo Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 We strongly encour
30. eral consecutive samples shift click the first and last sample in the group you wish to select 2 Choose Standard Sizing Curve from the Project menu Figure 9 E EES Sample File 0 Size Curve S5555 Best Fit 2nd Order Curve AO 2 92353552E 01 Al 9 240988E 02 A2 1 290388E 07 R 2 1 000 Align By Size Curve Best Fit 2nd Order Curve Size Calling Curve Local Southern Method 1 400 zl 00 2800 3500 4200 4800 5600 Data Point Figure 9 Standard Sizing Curve window The 2 value and the coefficients of the curve are provided The R 2 value is a measure of the accuracy of fit of the best fit second order curve Note You can only display the sizing curve for a sample if a valid sizing curve exists for that sample 3 Examine how the data points fit on the curve and look at the R 2 value to evaluate the size calling The data points should fit close to the curve and the R 2 value should be between 0 99 and 1 00 4 When you are finished click the close box 27 Defining Polymorphic Peaks for Genotyper Analysis In addition to sizing AFLP fragments GeneScan software enables you to prepare AFLP results data for downstream analysis by the Genotyper software application Before starting Genotyper define the polymorphic peaks to be scored Step Action 1 In GeneScan overlap the analyses of reactions from different samples to identify the polymorphic peaks 2 Under the View menu use the Custom Colors option to change
31. esting New Genomes 0 0 00 cee eee eee eee 6 Primer Selection Guidelines eee cee eee eee eee 7 Genome Analysis Guide 0 0 8 Fluorescent Dye labeling and Marker Detection 9 Materials Needed to Perform AFLP 0 0 0 0 cece eee eee eee 10 AFLP Kit Modules se rede Ge Heelan SER 10 AFLP EcoRI Ligation Amplification Module eee eee 10 AFLP Msel Ligation Amplification Module eens 11 AFLP Amplification Core Mix Module lesse 11 Storage and Stability of Kit Components 11 Materials Required But Not Supplied 20 5 12 Sample Preparation ee eee 14 Before Starting an AFLP Experiment 14 Preparing Samples for PCR Amplification 14 Anneal Adaptor 213 15 Prepare Enzyme Master eee eee 15 Prepare Restriction Ligation 62060023 16 Dilute Restriction Ligation 62060103 16 Amplification of Target 65 eee eee eee eee 17 OVEIVIEW oot hehehe ed eek e LEE Ue heb 17 Preselective Amplification 0 0 0 cece eee eee eee ene 17 Verify Successful Amplification 18 Prepare Templates arre
32. ethod shown in Table 3 entering all ramp times as 0 01 one second on the GeneAmp PCR System 9600 and DNA Thermal Cycler 480 or 9096 on the GeneAmp PCR System 2400 4 Store at 2 6 C Table 3 Thermal cycler parameters for preselective amplification CYCLE HOLD Each of 20 Cycles HOLD 72 C 94 C 56 C 72 C 4 C 2 min 20 sec 30 sec 2 min forever continued on next page 17 18 Verify Successful Run an agarose yield gel to check that amplification has occurred Amplification Step Action 1 Run 10 uL of each reaction on a 1 5 agarose gel in 1X TBE buffer at 4V cm for 3 4 hours 2 Stain the gel with ethidium bromide WARNING Ethidium bromide is a powerful mutagen and is moderately toxic Wear gloves a lab coat and safety glasses when using this dye 3 View the gel on a UV transilluminator A smear of product from 100 1500 bp should be clearly visible Figure 6 right half 1 ug of Undigested DNA Preselective amplification products 10 uL lane create a visible smear in the 100 1500 bp range Bst Ell of DNA size Boehringer Mannheim standards 1 2 7 2 kb DNA MW markers set V 1 ug of EcoRI digest j 3 Nic w 3i 3 ez gt z Am Bst Ell of A 1 ug of DNA after EcoRI and Msel 124 size standards digests These sequences are amplified zs Figure 6 Gel results after restriction digestion of 1 3 ug of DNA left a
33. gments generating template DNA for subsequent polymerase chain reaction PCR amplification Restriction and ligation may take place in a single reaction if the buffers are compatible Figure 2 Adaptor sequences have been designed such that ligation of the adaptor oligonucleotide to the restricted DNA does not regenerate the recognition site If the buffers are not compatible the reactions must be run sequentially A Cut genomic DNA into fragments with the restriction enzymes Msel and EcoRI FU M pM jh B Ligate adaptors EcoRI E 2 0 Msel C Modify genomic DNA fragments Msel Msel EcoRI EcoRI Figure 2 Example of template preparation and AFLP adaptor ligation The sequences of the adaptors and the restriction site serve as primer binding sites for a subsequent low level selection or preselective amplification of the restriction fragments Only those genomic fragments that have an adaptor on each end amplify exponentially during PCR amplification Figure 3 on page 4 This step effectively purifies the target away from sequences that amplify only linearly i e those with one modified end Prepared template genomic DNA fragment modified with adaptors Hr Preselective primers Bl Econ adaptor recognition site Adaptors Thermal Core Mix Cycling EE Figure 3 Preselective amplification of the prepared
34. ith GeneScane Analysis and Genotyper software Materials Needed to Perform AFLP AFLP Kit Modules The AFLP Microbial Fingerprinting Kit P N 402948 is organized into 10 AFLP EcoRI Ligation Amplification Module three individual modules AFLP EcoRI Ligation Amplification Module P N 402941 AFLP Msel Ligation Amplification Module P N 402942 AFLP Amplification Core Mix Module P N 402005 The AFLP EcoRI and Msel Ligation Amplification Modules provide sufficient reagents to modify and do preselective amplifications on 100 individual DNA samples 10 ng each There are sufficient amounts of the 18 selective primers nine EcoRI and nine Msel to test two preselective samples with all 81 selective primer pair combinations and to test the remaining 98 samples with six selective primer pair combinations The AFLP Amplification Core Mix Module contains all of the components necessary to amplify modified target sequences This module P N 402941 contains the following reagents EcoRI adaptor 100 uL 2 uM EcoRI core sequence 50 uL 10 uM Nine selective primers each 500 uL 1 uM Selective primer Designation AFLP EcoRI 0 FAM no additional nucleotide 0 AFLP EcoRI A FAM 1 AFLP EcoRI C NED 1 AFLP EcoRI G JOE 1 AFLP EcoRI T JOE 1 AFLP EcoRI AA JOE 2 AFLP EcoRI AC FAM 2 AFLP EcoRI AG JOE 2 AFLP EcoRI AT NED 2 E coli W3110 Reference DNA 25 uL 10 ng uL sufficient for 25 restriction
35. ligation reactions continued on next page AFLP MseI Ligation Amplification Module AFLP Amplification Core Mix Module Storage and Stability of Kit Components This module P N 402942 contains the following reagents Msel adaptor 100 uL 20 uM Msel core sequence 50 uL 10 uM Nine selective primers each 500 uL 5 uM primer Selective primer Designation AFLP Msel 0 no additional nucleotide 0 AFLP Msel A 1 AFLP Msel C 1 AFLP Msel G 1 AFLP Msel T 1 AFLP Msel CA 2 AFLP Msel CC 2 AFLP Msel CG 2 AFLP Msel CT 2 E coli W3110 Reference DNA 25 uL 10 ng uL sufficient for 25 restriction ligation reactions This module P N 402005 provides five tubes of Core Mix total volume 7 5 mL containing the following buffer nucleotides AmpliTaqe DNA Polymerase The AFLP Amplification Core Mix Module contains sufficient reagents for 1000 amplification reactions 10 uL each of target genomic sequences You determine how the selection occurs by choosing primer pairs from the AFLP EcoRI and Msel Ligation Amplification Modules Note For information on ordering selective primers and other AFLP reagents individually see Appendix C on page 38 Store all kit components at 15 to 25 C in a non frost free freezer If stored properly kit components will last up to one year continued on next page 11 Materials Reagents see Appendix C on page 38 for more information Required But Not W
36. lysis Range r size Call Range Full Range I All Sizes m This Range Data Points C This Range Base Pairs Start Min D Stop Max Data Processing r 5ize Calling Method Ex Baseline D 2nd Order Least Squares Ed Multicomponent 3 3rd Order Least Squares Smooth Options Cubic Spline Interpolation C None Oo p p amp Light Heavy Local Southern Method C Global Southern Method Peak Detection Split Peak Correction Peak Amplitude Thresholds Cj None O GENESCAN 2500 LettMast Peak RightMost Peak Min Peak Half Width Pts Correction Limit S0 Data Pts Figure 7 Analysis Parameters dialog box on the ABI PRISM 377 Setting GeneScan Analysis Software Parameters continued Step Action 2 Click OK 3 In the Analysis Control Window define a size standard as follows a Indicate the dye color of the Size Standard b Choose Define New from the pop up window and select a Sample File data for one lane The size standard peaks within the defined Analysis Range appear c Assign a size value to each peak d Close the window and enter a standard name when a prompt appears 4 Highlight the sample s to be analyzed and click on the Analyze button 5 After a successful analysis view your results in the Results Display window and then save the project 6 Select Save As from the File menu to save the data to a file
37. mber c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request through the Internet for fax or e mail delivery a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery 47 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trai
38. must anneal the adaptor pairs supplied with the AFLP EcoRI and Pairs Msel Ligation Amplification Modules before you can use them for the restriction ligation reactions Step Action 1 From the AFLP EcoRI and Msel Ligation Amplification Modules remove the tubes labeled Msel Adaptor Pair and EcoRI Adaptor Pair Heat tubes in a water bath at 95 C for five minutes Allow tubes to cool to ambient temperature for ten minutes Spin in a microcentrifuge for ten seconds at 1400 x g maximum Prepare Enzyme Prepare an Enzyme Master Mix to perform the restriction ligation Master Mix reactions for all 100 DNA samples or a proportionate amount for fewer reactions Step Action 1 Combine the following in a sterile 0 5 mL microcentrifuge tube 10 uL 10X T4 DNA ligase buffer with ATPa 10 uL 0 5 M NaCl 100 Units Msel 500 Units EcoRI 100 Units T4 DNA Ligase Add sterile distilled water to bring the total volume to 100 uL Mix gently Spin down in a microcentrifuge for 10 seconds Store on ice until ready to aliquot into individual reaction tubes IMPORTANT For best results use the Enzyme Master Mix within one to two hours Do not store Master Mix beyond the day on which itis to be used a 1X T4 DNA Ligase Buffer with ATP 50mM Tris HCl pH 7 8 10 mM MgCl 10 mM dithiothreitol 1 mM ATP 25 ug ml bovine serum albumin continue
39. nd after preselective amplification right continued on next page Prepare Template Prepare the preselective amplification products for selective amplification Step Action 1 Combine the following in a sterile 0 5 mL microcentrifuge tube 10 0 uL preselective amplification product 190 0 uL TE s buffer 2 Mix thoroughly then spin down in a microcentrifuge for ten seconds 3 Store the diluted preselective amplification product at 2 6 C if not used immediately Selective Amplify the EcoRI and Msel modified fragments Amplification Step Action 1 Combine the following in a PCR tube 0 2 mL for the GeneAmp PCR System 9600 or 2400 0 5 mL for the DNA Thermal Cycler 480 1 5 uL diluted preselective amplification product 0 5 uL Msel primer at 5 uM 0 5 uL dye labeled EcoRI primer at 1 uM 7 5 uL AFLP Core Amplification Mix Note If using the DNA Thermal Cycler 480 add 20 uL of light mineral oil to the tube 2 Run PCR using the thermal cycler parameters shown in Table 4 on page 20 Note For the GeneAmp PCR System 9600 and DNA Thermal Cycler 480 enter all ramp times as 0 01 one second For the GeneAmp PCR System 2400 enter all ramp times as 9096 3 Store at 2 6 C 19 Table 4 Thermal cycler parameters for selective amplification Number of HOLD CYCLE Cycles 94 C 94 C 66 C 72 C 1 2 min 20 sec 30 sec 2 min 94 C 65 C 72 C 1 20 s
40. ned support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com Applied E Biosystems Applied Biosystems is committed to providing the world s leading technology and information for life scientists Printed in the USA 06 2010 Part Number 402977 Rev F
41. ogy 142 1881 1893 Lin J J Kuo J Saunders J A Beard H S MacDonald M H Kenworthy W Ude G N and Matthews B F 1996 Identification of molecular markers in soybean comparing RFLP RAPD and AFLP DNA mapping techniques Plant Molecular Biology Reporter 14 156 169 Meksem K Leister D Peleman J Zabeau M Salamini F and Gebhardt C 1995 A high resolution map of the R1 locus on chromosome V of potato based on RFLP and AFLP markers Molecular and General Genetics 249 74 81 Money T Reader S Qu L J Dunford R P and Moore G 1996 AFLP based mRNA fingerprinting Nucleic Acids Research 24 2616 2617 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Press NY Thomas C M Vos Zabeau M Jones D A Norcott A Chadwick B and Jones J D G 1995 Identification of amplified restriction fragment polymorphism AFLP markers tightly linked to the tomato Cf 9 gene for resistance to Cladosporum fulvum The Plant Journal 8 785 794 Valsangiacomo C Baggi F Gaia V Balmelli T Peduzzi R and Piffaretti J C 1995 Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies Journal of Clinical Microbiology 33 1716 1719 Van Eck H J Rouppe van der Voort J Draaistra J van Zandwoort
42. ons of AFLP The AFLP amplified fragment polymorphism technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously The AFLP band patterns or fingerprints can be used for many purposes such as monitoring the identity of an isolate or the degree of similarity among isolates Polymorphisms in band patterns map to specific loci allowing the individuals to be genotyped or differentiated based on the alleles they carry AFLP technology combines the power of restriction fragment length polymorphism RFLP with the flexibility of PCR based technology by ligating primer recognition sequences adaptors to the restricted DNA Some of the advantages of the AFLP technique are the following Only small amounts of DNA are needed Unlike randomly amplified polymorphic DNAs RAPDs that use multiple arbitrary primers and lead to unreliable results the AFLP technique uses only two primers and gives reproducible results Many restriction fragment subsets can be amplified by changing the nucleotide extensions on the adaptor sequences Hundreds of markers can be generated reliably High resolution is obtained because of the stringent PCR conditions The AFLP technique works on a variety of genomic DNA samples No prior knowledge of the genomic sequence is required Applications for AFLP in microbial fingerprinting include the following differentiation and tracking of highly related microbes at the
43. rs Cosmids BACs 0 0 EcoRI 0 FAM Msel 0 P1 mapping YACs some 0 1 EcoRI 0 FAM Msel A larger BACs Msel C Msel G Msel T 1 0 EcoRI A FAM Msel 0 EcoRI C NED EcoRI G JOE EcoRI T JOE Bacteria 0 2 EcoRI 0 FAM Msel CA Msel CC Msel CG Msel CT 1 1 EcoRI A FAM Msel A EcoRI C NED Msel C EcoRI G JOE Msel G EcoRI T JOE Msel T 2 0 EcoRI AA JOE Msel 0 EcoRI AC FAM EcoRI AG JOE EcoRI AT NED Yeast small fungi 2 2 EcoRI AA JOE Msel CA genomes EcoRI AC FAM Msel CC EcoRI AG JOE Msel CG EcoRI AT NED Msel CT Large fungi 2 3 Use the primers from the AFLP Regular genomes 3 2 and Small Plant Genome Mapping Kits See Appendix C on page 38 for the primers available continued on next page Table 2 Genome Analysis Some bacterial and fungal genomes that have been analyzed Guide successfully using EcoRI Msel and the primers in this kit are shown in Table 2 Genomes analyzed with EcoRI and Msel primer pairs Primer Pairs Used Organism Successfully Primer Pairs to Avoid gt Acinetobacter sp EcoRI C Msel T Aeromonas sp EcoRI A Msel T Aspergillus sp EcoRI A Msel G EcoRI A Msel CA EcoRI C Msel CA EcoRI T Msel A Bacillus sp EcoRI 0 Msel A Candida utilis Clostridium sp Vancomycin resistant Enterobacter Escherichia coli Eutypa sp Legionella pneumophila Nensenula anomola Paenibacillus larvae Pichia membrefaciens Saccharomyces sp Schizosaccharomyces pombe Xanthomonas sp EcoRI
44. s including but not limited to production activities commercial activities and any activities for the commercial benefit of third parties for or in connection with but not limited to plant breeding seed quality control animal genetic testing or breeding microbial typing human diagnostics human genetic testing human identity testing or human disease testing requires a license from Keygene N V P O Box 216 6700 AE Wageningen The Netherlands ABI PRISM Applied Biosystems GeneScan Genotyper and MicroAmp are registered trademarks and AB Design and Applera are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpliTaq and GeneAmp are registered trademarks of Roche Molecular Systems Inc AFLP is a registered trademark of Keygene N V All other trademarks are the sole property of their respective owners P N 402977 Rev F Contents Introduction 0 Db ei ee Wee ee es 1 Whatas AFLP sess sae pene trol heds tae sale ae RAS 1 Advantages Of AFLP essei RR ey rg ea aes 1 Applications of AFLP 1 0 0 0 cee eee tenes 1 The AFEP Technique Lose to dr HERPES reed de bie de MESES 3 Template Preparation and Adaptor Ligation 3 Preselective Amplification 3 Selective Amplification carie eee 5 Simplifying Complex Patterns 5 T
45. see page 19 or the modified restriction fragments can be amplified in preselective AFLP reactions first see page 17 then amplified selectively How to choose what reactions to perform is discussed in The AFLP Technique on page 3 Amplification of Target Sequences Overview This protocol has been optimized for the GeneAmp PCR Systems 9600 and 2400 and the DNA Thermal Cycler 480 If you use a different thermal cycler you may need to optimize the conditions The temperature ramp times included in this protocol ensure identical products from any Applied Biosystems thermal cycler Ramp time is crucial If the temperature is increased too quickly results may be inconsistent See Appendix A on page 32 for troubleshooting tips Preselective Sequences with adaptors ligated to both ends amplify exponentially and Amplification predominate in the final product Note Keep all reagents and tubes on ice until loaded into the thermal cycler Step Action 1 Combine the following in a PCR reaction tube 0 2 mL for the GeneAmp PCR System 9600 or 2400 0 5 mL for the DNA Thermal Cycler 480 4 0 uL diluted DNA prepared by restriction ligation 0 5 uL AFLP EcoRI preselective primer 0 5 uL AFLP Msel preselective primer 15 0 uL AFLP Amplification Core Mix Note If using the DNA Thermal Cycler 480 overlay your samples with 20 uL of light mineral oil Place the samples in a thermal cycler at ambient temperature Run the PCR m
46. template Msel adaptor recognition site In the microbial genomes targeted by this kit the core primer sequence is used In larger genomes such as plants and some fungi this amplification would create too many fragments In those cases the preselective amplification is performed with additional nucleotides on the end of each primer Each added nucleotide reduces the number of sequences by a factor of four The thermal cycling conditions of the preselective amplification step have been optimized to generate a constant final mass of fragments Band intensity in subsequent reactions can therefore be correlated with relative differences in representation of the fragments within the genome and not to the overall amount of genomic DNA that went into the initial restriction ligation mix It is not necessary to perform this step if relative peak height information is not desired methods are available to normalize the final signal very accurate quantitation of the input DNA is performed routinely continued on next page Selective Additional PCR amplifications are run to reduce the complexity of the Amplification Simplifying Complex Patterns mixture further so that the fragments can be resolved on a polyacrylamide gel These amplifications use primers chosen from the 18 available AFLP Microbial Fingerprinting Kit Selective Primers nine EcoRI fluorescent dye labeled primers and nine unlabeled Msel primers After PC
47. urs ABI PRISM 377 36 cm 2500 volts 4 0 hours continued on next page Preparing the Loading Buffer for the ABI PRISM 310 Loading and Electrophoresis on the ABI PRISM 310 Prepare a loading buffer mix of the following reagents in the proportions shown in sufficient quantity for each sample 24 0 uL deionized formamide 1 0 uL of GeneScan 500 ROX size standard WARNING Chemical hazard formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use chemical resistant gloves and safety glasses when handling Note You can store any remaining loading buffer at 2 6 C for a week For specific instructions about loading and running samples refer to the ABI PRISM 310 Genetic Analyzer User s Manual Step Action 1 Add 25 0 uL of the loading buffer mix to a sample tube Use one tube for each sample Add 0 5 uL of the selective amplification product to the tubes Heat tubes to 95 C for 3 5 minutes Quick chill on ice Place the Genetic Analyzer sample tubes in the 48 well or 96 well sample tray a Use 0 5 mL Genetic Analyzer sample tubes for the 48 well sample tray and 0 2 mL MicroAmp Reaction Tubes for the 96 well sample tray IMPORTANT Use the GS STR POP4 A run module and ABI PRISM 310 Genetic Analyzer Collection Software version 1 0 2 or higher with the AFLP Microbial Fingerprinting Kit Make the matri
48. x with the Dye Primer Matrix Standards P N 401114 substituting the NED Matrix Standard P N 402996 for TAMRA Table 6 ABI PRISM 310 Electrophoresis Parameters Pattern Injection Time Injection Run Time Run Voltage Complexity sec Voltage kV min kV Dense patternsa 12 15 30 13 Simple patterns 5 13 26 15 a Use these conditions when running any sample for the first time continued on next page 23 Using GeneScan to After your sample data is collected you can use GeneScan Analysis Analyze Results software to analyze and display sizing results for all samples in any combination of tabular data and electropherograms with or without legends When you display electropherograms and tabular data together the Results Display window is divided into upper and lower panes The upper pane contains electropherogram panels and the corresponding legends the lower pane contains the tabular data The following procedure describes how to set the GeneScan Analysis software parameters For more complete information refer to the AB PRISM GeneScan Analysis Software User s Manual Setting GeneScan Analysis Software Parameters Step Action 1 Under the Settings menu select Analysis Parameters Set the parameters as shown in Figure 7 for the ABI 373 and ABI PRISM 377 On the ABI PRISM 310 use an analysis range of 2600 10000 data points and peak amplitude thresholds of 100 Rnalysis Parameters Rna
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