AFLP® Plant Mapping
Contents
1. The AFLP Ligaton and Preselective Amplification Module contains sufficient reagents to prepare an intial mapping population ot up to 100 individual For the tasting of each addtional 100 individuals in a nou AFLP Ligation and Preselecive The AFLP Ampilication Core Mix Mode supplies sufficient PCR mixto pez 500 individual AFLP reactions AFLP Ligation Amplification The AFLP Selective Amplification Start Up Module supplies suficient quantities of primers to test al 64 possible primer combinations on 30 individuals chosen from the 100 individuals prepared withthe AFLP Ligation and Preselctve Ampliication Modue For each primer combination you can compare 4 the total number of peaks amplified in the parents 4 the number of polymorphic peaks between the parents 4 the segregation ratios of polymorphic peaks in progeny of the cross Once you estabiish the most useful primer combinations for your sepies you can purchase 250 or 500 reactions of primer along with he AFLP Ampileston Core Mix Module The Core Mix Module contains the necessary reagents for performing PCR The primer combination tables in Appendix A on page 38 show primer combinations best sulted for analysis of tn dierent major crop Species You can order those primers soparaaly eee pagos 7 6 Template preparation and preselective amplification require use of the AFLP Ligation and Preeelectve Amplification Module Regular Plant Ge2. Gan CAC CAG CAT Len cT cra len me asa O 8 ESS 5 aco aca 8 8 sac pa 8 Table 15 Primer combinations for Arabidopsis species s ama genome can CAC cAa CAT CTA cre er E D H o ac Table 16 Primer combinations for Cucumber species ema genome eu CAC CAG CAT CTA cre Lem crr sc O NOT DETERMINED Table 17 Primer combinations fr Rice species mal genome eu CAC CAG car cra ere cra e ES xa Appendix B Troubleshooting Table 18 Toubloshooting AFLP procedures ET Possible Causes SE ba eer apt meroon yaa Win tra enzymes and bute Use an pit check Pon ios may sine DNA sange Ty aera Bran OOS Use an aparece galt hack EI ak N DNA sued nwa hot water pa Tes ray Say rostcson gasten enzyne gaas and Apa DNA Polymerase Eidel EER ed Saan o Green een Remake th Teo EI parameters TE SEE SST ln parameter Debt ge che ee e me GT EE Bo ea GR Ph an ba o omic in Hoer betore frat yes Wong aye e Ed DNA Mermal Gye ie o rom Reston Tubos win Captor mne Gnaamp PCR System 8000 or System 2400 Ve recommended privar II EEES Table 18 Troubleshoo
3. Figure 1 Tomato AFLP sangla shoning Mendetan sepagatan The overlapping electropherograms in the top panel are AFLP results of sample DNA from three individuals parent one P1 parant wo P2 and FI from a cross A and B ara tha two significant peaks on this panel and appear ony in P2 and FI The lower three slectropherogram panes are AFLP resus of sample DNA from tres F2 generations Peak A appears in F2 8 but does not appear in ether F2 1 or F2 2 Peak Bls inherited in all threo F2 individuals The remaining non polymorphic peaks appear in al three F elocrophorograms and show thatthe overall AFLP pattems are reproductie Figure Res AFLP sagas snowing nea oganio regions The two elecropherogram panels shown in Figura 2 contain data from ros DNA samples prepared using the AFLP technique Samples wore fun on an ABLS73 DNA Sequencer and the resulig data analyzed using GenaScan Analysis sofware The rice DNA was isolated trom nearisogeni ines almost identical genetic materia was selected for an ntogrecsed reglon carrying a seso resistance gone By comparing peak patterns i the wo alctopherograms you wil find that the rc Iines ie by only 1 29 One ofthe peaks distinguishing th two Ines has been highlighted in both the alocropharogram display and iho related tabular data beneath the electopherogram panels The AFLP Technique Template The rst stop of the AFLP technique is to generate restric
4. DNA Sequencer and ABI Presu 310 Genee Analyzer use Virtual Fater Sar For the ABI Pris 377 Fiter Set F modulo fles can be obtained from he Applied Blosystoms World Wide Web ste as part of the ABI Pato 977 Colection sofware version 21 wwwapplodbiosystoms comechsuppert ABL PRISM 877 va 1 image he For the ABI Pris 310 Fiter Set F modulo fles will be part of the next release of the ABI PRISM 310 Colection software version 1 0 4 Prepare a loading bute mico the folowing reagents in the proportons shown in suficient quantiy for each sample 1251k deionized formamide 0 25 uL blye dextran 25 mM EDTA loading solution supplied wit the sizs standard 4 OS ul of GeneScan 500 ROX size standard WARNING 1 Chemical hazard formamide is a teratogen and ie harmful by inhalation skin contac and ingesien Use n a weil ventisted area Use chemleeesatat loves and ally gastes when handing Note You can store any remaining ang butler a126 O lor 1 week Loading and Electrophoresis on the ABI 373 and ABI Prism 377 For specific instructions about loading and running samples rafer to tho ABI 373 DNA Sequencing System Users Manual or he ABI PAIS 377 DNA Sequencer Users Manual E he ABT E Sequencer Sequencer RASS M oe dig Ba AGI Zo re Ed AE POR uber acn sample Zoe 7 raros es TEL Ze aneneen oracio ne EN in ene lanm add GERS each eaten reke EL er Fee GE Cr NL Eer
5. Displaying the Standard Sizing Curve EI Selec a sample Gr muli samples Pe Ane Core To soc several consecuve same sil eta and last sample in the group you wah select Chaos Sas Cave ron Fa Sample wa The SST Sang The vlan econo the curve are provided The Fre a i s measure te accuracy of of o boa second Note You can oniy daga the sizing curve tor a sample a vait ating curve ent fo a sale anne how e dala pais on Gave ad wok aha PE Veo evaluate these ang Ta points hk kwe o ecu ana te Fae Tan your Wished che doe bak Deining Polymorphic Peaks for Genotyper Analysis In aaaton to sizing AFLP fragments GenaScan software enables you to prepare AFLP resus data for downstream analysis by the Genotyper software application Before starting Genotyper define the polymorphic peaks to bo scored E EES SES Ng aras lesions From dean Sangla o erty he palman Peaks Under a View menu use Te Custos CT aN e anga The play color ot ane or more ot he samples so na he Ee FO eco mo sas ofthe polos paka and ng samples Par produced er Figure 8 shows the polymorphic peak pattems rom a GeneSean analysis of two AFLP samples Polymorphic peaks ar labeled wih size and origin Figures Overlapping electophrograms tor two AFLP samples You can import GeneScan resul data into a Genotyper software template Used together GeneScan and Ganotyper can avtomate segregat
6. Eck Hul Rouppe van der Voort J Draaistra J van Zandwoort P van Enckevort E Segers B Peleman J Jacobsen E Helder J and Bakker J 1995 Tho inhertanc and chromosomal location of AFLP markers in a non inbred potato offspring Molecular Breeding Lara Vos P Hogers R Blekor M Rejans M van de Lee T Homes M Fira A Pot J Peleman J Kuiper M and Zabeau M 1995 AFLP a now concept fr DNA fingerprinting Nucl Acids Ras 28 4407 4414 Zabeau M and Vos P 1998 Selective restriction fragment mplfcaton a general method fr DNA ingerprinting European Patent Application EP 0534888 Appendix E Related Consumables and Accessories This appendix contains ordering information and descriptions of different kits and consumables which you can use to parorm procedures described in this protocol Table 19 Related consumables and accessories pas Lee a Sen S Ee EES seas EE a Sse A EAS E a ES as a S DES Desen a ree DT EE meree rasta SES Leen N Se RESET Te as sere quemar AE ER D lege EE Som Tee E Se P277 Sco Table 19 Related consumables and accessories eontu Ge es Ganang pore Ed Fann PWOFITE ses Gagana labi ona anjo sand van ROK NH energy Shippea in wo ubos containing 20 al mete esch Se ners between 5 Ewe Dye Pier Nati Standard Rt bog FAM
7. JOE and FOX crocs sera wavelengths tee l ome overap nthe meo peca To Conect tor s ota Ko een he appropriate mae pied ee dao act out ary emission Ko pad Bayan TED Wai Sad ee sb HED e yelow dye nthe AFLP Para Mapping Ki ees wurwappliedbiosystems com AB Stems
8. 0 on page 40 Mize Table 11 on page 40 Sugar bet Table 12on page at Tomato able 13 on page at Larue abo 14 on page a2 mel Plant Genomes Cen Table 15 on page a2 ee abo 16 on pages Res Table 17 on pages The folowing symbol indicates unacceptable primer combinations for amplfeation sereening of designated speck Table 8 Primer combinations for Sunflower species eu CAC caa car ora cre e 8 8 Se eof Primers e BEA AE J l o 8 8 Table 8 Primer combinations for Pepper species CAA CAC CAG CAT en o o 8 ol e 9 EcoRI Primers Ble a EE EIE Table 10 Primer combinations fo Barley species can CAC caa CAT ora Les cra len aa Se E ea pen en scr O H o Table 11 Primer combinations for Maize species 5 5 JE IE o o ou H Table 12 Primer combinations fr Sugar beet species eu CAC caa car ora cre Serge l l El Table 13 Primer combinations for Tomato species eu CAC CAG CAT CTA oro eof Priors l AE EE Elk Table 14 Primer combinations for Lettuce species
9. 1 precede the country codo and ave tue ou Each document in and has an 10 number Ue sa your order number in top balm a Cal 1258 7120017 trom auctions phone a 4 Pros 2 1o order up to ve documents and have them tied you o Reach Us by Contact technical support by e mail fr hap in the folowing product E Mail areas or this product area TIETE Er FEESTE ST Gee Aras II TERE EETL GEEIS IE nee tala Sequencing EE Bappiedhbaysamacom NA Sis Regional Offices 1 you ar outside the United States and Canada you should contact Sales and Service Your local Appled Biosystems service representatie ned See Tan Aaa DAA Obregon AE Mao 285 Leon Gerre Dre Fostar ly Cala gasoa Teh passe rd Fax POSS bus groe Fax Coso Europa asia Wen Tanga Guias Tai 430186735750 ENE Oo ELE GE Tay ano Ta mens Tet moin Fan s2 a712 s518 Foc Seene Goch Rapa md Svat The Nan Neuve a ana busa Teh 420261222104 Tet o 08051400 Fax 40201 222 68 TE Denmark aeran Norway 050 E Friend Eo Tet asa a251 24250 Fax asa a251 24203 oid WERE Lav and Estonia warszawa Tai 48 22 80640 10 Fan B122 aco 4020 anos aray Tek nmn Far Sunnen EI Tet 381 00226053914 Fax 951 0022605 2015 Germany tese Te e ersa 1010 Fax 49 0 6150101 101 a ose Spain Tes Cane Tae 3481808 1210 Fax ges Sout Aa ohana Sweden Sean Tek
10. AFLP Plant Mapping Protocol AB BEE seme cop SI aga Rn a eA Me th ala o ma pu pes Saf sy ss put e Te pt pda rt pra al aN nays psu de a acne Pee bakong to par ena spo mer rey cy wa ian og pn fsa ar some onsen fa Sto eye Te ich oh Tr mt o prang vn ay od EECH Te AFLP pss ol a pu ops EEN Ths i io an Shame a kn e Sci ngng ri am phan a at or A RISA iS May Con Gya Mi ay pil o AB o 8 Api vant ype Ba nr a US ano cnica plan e aro von Rc Mac pal Contents ofan What is AFLP Advantages of AFLP Applications of AFLP The AFLP Technique Template Preparation and Adaptor Ligation eene Amplification Selective Amplification Choosing Spec Primers for Amplcaon Screening Tesing New Genomes Dem Dye heling and Marker Detection Technical Support To Reach Us on the Web Hours for Telephone Techaical Support To Reach Us by Telephone or Fax in North America Documents on Demand To Reach Us by E Mail Regional Os Sale and Service What You Wil Need to Perform AFLP Overview AFLP Kit Modules AFLP Ligation and Preselective Amplification Module AFLP Amplification Core Mix Module AFLP Selective Amplification Start Up Module Storage and Stability of Kit Components Noel Required But Not Supplied AFLP Pia Mapping Protocol a B D 16 16 a7 18 18 18 w 20 Before Starting an AFLP Experiment Preparing Samples for PCR Amplia Annealing Adaptor Pais Preparing Bazyme Master Mix Preparing
11. ON Sa LOG Ed moa dang St dena Long Paner gel BEE 1X TBE rurning tr fat ing baa IMPORTANT Use Finer Sat Ai ABI 373 and Fer Set Fw be AB Pris377 DNA Sequence when anang samples prepared wih he AFLP Piani Mapping KA modes see un Mos on paga 27 ako he meri win a Bye Primer Max Standards PIN 401114 abating De NED Mat Standard PIN 402308 TAMRA Table 5 ABI 373 and ABI PRISM 977 Electrophoresis Parameters Ting parametar Time Preparing the Loading Buffer for the ABI Pusu 310 Loading and Electrophoresis on the ABI Pit 310 Prepare a loading butler mik the folowing reagents in the proportons sown in suficient quantiy for each sample 240 uL deionized formamide 10 uL of GeneScan 500 RON size standard WARNING 1 Chemical hazard formamide le a teratogen and ie harmful inhalation akin contact and Ingestion Usain a weti een ate Uae cremea resistant gloves and deer when handing Note You can store any remaining ang butlar at 2 6 G Y wash For specific instructions about loading and running samples rafer to the ABI Prisi 310 Genes Analyzer Users Manual CAL EET dog iar io a sampl bo Usa ara befor sach sample AA S pL he sls Ap proa Pe baa Fee EE CET Galeton ee Pare De Genote Aral yasr ETE WEE Re TE Re Gr BE we sangat Use OL Ged ajar anga a KG slay OAL Merk Rea ee ml sale ay IMPORTANT Use the GS STR POPA F un module and A Pr Banat Analyzer C
12. Rosie Ligation Reactions Ding Resriction Ligation Reactions Amplias of Tape Sequences Overview EE Venjing Successful Steg Preparing Template Selective Amplification rang Resales Overview Ran Modules Preparing the Loading Buller for the ABI 373 and ABI Pais 377 Loading and Electrophoresis on the ABI 373 and ABI Pins 377 Preparing the Loading Buffer for Be ABI Pais 310 Loading and Electrophoresis on the ABI Psx 310 Using GeneScan o Analyze Results Bvalusting ABI373 DNA Sequence Results Bvalusting ABI PRIM 377 DNA Sequencer Resuli Evaluating ABI Pats 310 Genetic Analyzer Results Appendix A Primer Conbinaton Tables Genomes Analyzed Uring AFLP Appendix B Troubleshooting Appendix C Preparing Plant Genomic DNA Appendix D References Appendix E Related Consumables and Accessories ki sbvEESkk bkREBE kekke Introduction What is AFLP Advantages var Applications A AFLP The AFLP amplifed fragment polymorphism technique is used to visuals hundreds of ampiied DNA resticton fragments eech The AFLP band patios or fingerprints can be used for many purposes such as montoriag th deny of an isolata or the degree of smiariy among isolate Polymorphisms in band paterne map to speci loci allowing the indvduals to be genotyped or ierented based on the alleles they carry AFLP technology combines the power of restriction fragment length polymorphism RFLP with tha Got of PCR based technology by gating primer reco
13. Smal Plan Genomes PIN 4303051 with the Core Mix Module Each AFLP Selective Amplicaton StartUp Module contains 16 olgonuelsotde primers Tabie 1 on paga 7 This provides you with 64 possible combinations of primer pars that you can us in 30 reactions teach for a maximum of 2000 Selective Amplification reactions 4 Eight of the primers are complementary to the Mel adaptor sequence and have tree addtional bases at fhe 3 end Eight of the primers are complementary to the EcoRI adaptor sequence They have two PIN 4303051 or three PIN 4303050 tonal bases atthe 3 end and have 5 fuorescent dyes The primer ar labeled wih FAM blus JOE green or NED yelow Note Use tout color red ROX bran internal siza standard such as me Seneca 500 ROX Size Standard evade tom Applied Biosystems PN oras Once you determino optimal primar combinations you can purchase larger quanties 250 or 500 reaction equivalents of specie primer Combinations for testing of addtional DNA samples Store all kit components at 15 to 25 C in anon tostire freezer If stored property he kt components wil last 1 year fom tha time of receipt Materials Reagents ee Appendix E on page 50 fr more Information Required But Not 9 Supplied H Nuclease fee ditiled deionized water EcoRresticton endonuclease 500 Units high concentration grade Meal restriction endonuclease 100 Units high concentration grade T4 DNA Ligas
14. alecton Sohwar version 1 0 2 or higher vin ma AFLP Paring Ki mais ase Run Mad on page 27 Maka t matt wan he Dye Prenar Mat Standards PIN 401114 eben be NED Mate Standard PIN 42905 lt TAMRA Table 6 ABI Prou 310 Electrophoresis Parametors Panero Ster bat Run Time Ran Voge Sien Time see Voltage QV ei a Dense patea 12 ka E ka See ER BR See ER ay ao Be Re gr Using GeneSean to Afer your sample data is collected you can use GeneScan Analysis Analyze Results software to analyze and display sizing rau fo al samples in any Combination of tabular data and elachophorograms wih or without legende When you display elecropharagrame and tabular data together the Results Display window is died into upper and lower panes The upper pane contains elactopherogram panel andthe oresponcing legends he lower pane contains the tabular data The flowing procedure describes how to set the GeneScan Analysis software parameters For more complet information rater to the ABI Pris GaneScan Analysis Sofware Users Manual Setting GeneScan Analysis Software Parameters Sup Raton T Under Bi Sings ONG wee NSE PARTS SATS parametro ABI 373 and ABI Pr 377 a shown blow On he ABI Psa 310 so an anat range of 2800 10000 data Prins an peak pudo esol 100 pot Ed mg Oam arar tmn Samre Sp Fek Cree gamma ED Setting GeneScan Analys
15. e 100 Units high concentration grade 10X Ta DNA ligase buffer containing ATP Naci 05M nueease reo molecular biology grade Bovine serum albumin BSA 1 0 mg nucease ree AX TE a fer 20 mM Tie HEL 0 1 mM EDTA pH 8 0 nuclease free 6 denaturing polyacrylamide gel er the ABI 273 DNA Sequencer 5 Long Ranger gei for the ABI Paisu 377 DNA Sequencer Performance Optimized Polymer 4 POP 4 tor the ABI Prism 310 Genetic Analyzer Deionized formamide GeneScan 500 ROX Size Standard DNA size markers ag Boshringer Mannheim Set VI Dye Primer Matix Standard Kit NED Matrix Standard substtutes for TAMRA Segen 241 20 and 20041 with serie pote tps Gatioading pipette tips 0 17 mm fat or the ABI Pai 377 Applied Biosystems thermal cycler Sterle 0 5 mi microcentrituge tubes Sterle 0 2 mL MicroAmp Thin Walled Reaction Tubes and caps for the GeneAmp PCR Instrument Systeme 2400 and 9600 Store Thin Waled MicroAmp 0 5 m Reaction Tubes for ho DNA Thermal Cycler 480 AFLP Plant Mapping Protocol Before Starting an Before seting up an AFLP experiment you must first determine AFLP Experiment whether or not your genomic DNA restricts propery with EcoRI and Meel ms tte E EE EE TSE niare ge Foran example ot wat success gest ook ike see Figure n pag 24 ennan Preparing Samples IMPORTANT Betore you prepare your samples stony recommend leson relate joli e cad poss Ac
16. etection technology PCR products are dye labeled during ampiicaton using a 5 Greet primer For high throughput you can co lead up to three dierent Taactions labeled with dierent colored dyes ina sind lane on the ABI 379 or ABI Prusm 377 DNA Sequencer orin a single Injection on the ABI Prism 310 Genetic Analyzer Load an internal lane siz standard in a fourth color in every lane orinjectonto size al ampiicaton fragments accurately You can automate the scoring of the large numbers of markers that are pica generated by analyzing your results win GeneScan Analysis and Genatyper sofware Technical Support To Reach Us on the Web Hours for Telephone Technical Support To Reach Us by Telephone or Fax im North America Applied Biosystems wob site address is ip e appledbisystems comtechsupport We strongly encourage you to visit our web te for answers to requentiy asked questions and to learn more about our products You can aso order technical documents andor an index of mala Socuments and have them faxed or e mailed to you trough our ake see the Documents on Demand section below inthe United States and Canada technical supports avalabi at the folowing times Chemen TEE Esa Tine TONG EER GETOER Pac Tine Ge EER BEER Pas Tine See the Regional Offices Sales and Service section below for how o contact eal service representatives outside ofthe United States and Canada Cal Tactical S
17. grison sequences adaptors othe restricted DNA Some of the advantages ol he AFLP technique are the following Only small amounts of DNA aro needed 4 Unik randomly amped polymorphic DNAs RAPDs that use matpie arbitrary primers and lead to unreliable resuts the AFLP Technique uses only wo primers and gives reproducible resus Many restriction tagment subsets can be amplifed by changing the nucleotide extensions on the adaptor sequences Hundreds of markers can be generated reliably 4 High reslaton is obtained because of the stringent PCR Ze The AFLP technique works on a variety of genomic DNA samples 4 No prior knowledge of the genomic sequence is required Applications for AFLP in plant mapping include 4 establishing Image groups in crosses saturating regions o introgression with markers for gene landing forts 4 assessng the degree of relatedness or varabliy among culiar Examples of AFLP fingerprints are shown in Figure 1 on page 2 and Figure 2 on page 3 Literature references for tha AFLP technique are found in Appendix D on paga 48 meam rat ra re You can build a genetic map of markers showing Mendelan inhertance from AFLP data such as that shown in Figure 1 The four slectopherogram panels in Figure 1 contain data rom tomato DNA amples prepared using he AFLP technique Samples ware run on an ABI 373 DNA Sequencer and the resulting data analyzed using CGeneScan Analysis sofware
18. he mice so that it can be resolved on a polyacrylamide ge These lampiicatons use primers chosen trom the 24 avalable AFLP Selective Primere eight Mee and sixteen Ecol primers Ater PCR amplification with these primera a porton of each sample is analyzed an a Applied Biosystems DNA Sequencer Selective mplfcaton with an EcoRI and an Mel primer amplis primaiy EcoR Msel ondedragmonts The EcoRILECORIfragments do not amplify wol The Meet Meal fragments are not visualized because hey do not contain fuorescent dye labels Only the EooR contaning strands are detected Figure 9 A Crean Slee AFLP Pana AER Ace EE Gn Ke Finn he KM Ave on ina ansent o ca ant ug rn ELE Man St PR a Bee ie Figure Seale enplesian nn debes primers Individual genomes yield distinctive restriction fragment profs with ach primer pair amplification Those cop species genomes that have been analyzed successtuly using Meel and EcoRI and tha primers in ts kit are shown in Table 7 on page 38 Choosing Specific Primers for Amplification Screening if you want to use a specific primer combination forthe AFLP Selective Arer reactions you can order primer pairs in any combination of one EcoRI primer and one Mel primer This ges you 128 possible Primar pair combinations rom which you can choose for either regular smal plant genomes Order the AFLP Ampiiicaton Core Mit Module PIN 402006 and the desired AFLP Selective Ampl cato
19. ing Tho rosticton igaton reactions prepare the template for adaptors and Restriction hon ligate adaptor par to the prepared template DNA Ligation Reactions EAL MED EG 0 10X TA DNA Rose butter hat includes ATP DO 0544 10 maint BSA item 10 mpi necessary 1014 Mea adaptor DOC OU Enzyme Master Me NE neen ENE 0 05 yg genomie DNA SSL omg Rad 8 ES DNA TOT POT ram he AFLP Ugatonand Presectye Amputation Modu END an lace na micas or TO cards cuba at EE ortor Paas 37 C For inca eC use a thermal jr wth a hana cover ko mat re evaporation dass not laad to Eco a acy Be crt at me vozne of enzyme sadeg does not cause the ente gyesrd be 353 wich ao Ido to Eco acy Tiet Dilute the reticton igaton samples to va the appropriate Restriction concentration for subsequent PCR EAL E CRL Note Sore the mixture at 2 8 C tor up to 1 month ora isi 250 tor longer man 1 mon Amplification of Target Sequences Overview Tris protocol has been optimized forthe GensAmpo POR Systems 9600 and 2400 and the DNA Thermal Cycler 480 you use a diferent ermal cycler you may need to optimize the condition The ramp times included in this protocol ensure identical products rom any Applied Biosystems thermal cycler Ramp tine is crucial Se Appendix B on page 44 for troubleshooting tips Preselective Sequences wih adaptors gated to both ends amplify exponential
20. ion scoring of AFLP resis For mora information on how you can analyze polymorphic peaks using CGenatyper seo the Genotyper DNA Fragment Analysis Software Users Manual Evaluating you run samples under the recommended electrophoresis conditions ABI 373 DNA and analyze them with GeneScan rsulingelocrophorogram data Sequencer Results fem the ABI 373 DNA Sequencer shouid lock similar to data fom samples run on the ABI PRISM 377 DNA Sequencer Figure 9 shows a representative alactropherogram of fuorescent dyelabolod AFLP products run on an ABI 373 DNA Sequencer and analyzed using GeneScan analysis sofware Tho analyzed products are DNA fragments modi with Maa and JOE dye labeled EcoRI Selective ampuicaton primers The JOE labeled EcoRI fragments are splayed as peaks in the eleciropnerogram Evaluating Ant PRISM 377 DNA Sequencer representative clectopherogram of fuorescent dye laboled AFLP pat non an ABP dd GeneScan analysis software is shown in Figure 10 The produci are DNA rages amped vin ll and FAM ye ale Ecol selective ampifeaton primers Tho FAN abel EcoRI fragments are displayed as peaks inthe elecropherogram Figure 10 Etecrepherogam of AFLP sample run on an ABI Prisi 377 DNA H Figure 11 on page 37 shows an expanded elecropherogram of select peaks ram th samo AFLP samples shown in Figure 10 Tabular data in Figure 11 shows the sizes of sample fragments in mob
21. is temper crop ampere EI Sampe Seal nat Gana Comp Sampa Ghat cons Seen EEN Oo Cheah Wile concen ER matche protocol enmen epp Tersa rn pare Chea Log or o record ts een Appendix C Preparing Plant Genomic DNA Wil tho AFLP technique does not require as much genomic DNA as he RFLP technique the quality of the DNA is very important In articular the DNA must frat be restricted to completion wih enzymes nd then ged to adaptors betore the AFLP reactions are performed Tis appendix supples references for extraction and quaiiicaton methods fo preparing genomic plant DNA DNA Extraction Techniques Any particuiar plant species presents unique extraction problems so it is up to researchers to optimize a DNA extraction technique for their system Our sientas and those in many oer labe have had excellent results using the various CTAB purification schemes Doyle and Doyle 1000 For individual systems joumals such as Biotechniques contain numerous reports dealing modifications that improve the quality and or antiy af purihed DNA in various species including cotton and pine ex Bakar etal 1900 Quantating DNA Retar to molecular biology manuals such as Current Protocols in Molecular Biology for information on Quanttating ihe DNA restriction digestion procedures Pouring and loading geis Running and interpretation of agarose els Another good source of general infomation is Molecular Cloning A Laborator
22. is Software Parameters ued CAL z oase 3 mio Anaya Cora Window daine a Sis anda as blows a indicata to dje col of a Size Standard b Choose Dene Naw tom tho popup window and selecta Sami Fie ata or one and Tho size standard peaks wii the dead Anais Range pea Assign a size value to each peak A Close be window and enter a standard name when a prompt appear TGA NG TEE GE nad and KO NG BT D ser ven TEE RR WE HG Di dou ana hen sara ma project Ba Sa Ar hoe Fle man SG AA AG GeneScan S0 Sze Standard The GeneScan 500 standard is made of double stranded DNA fragments but only one ofthe stands is labeled with an ABI PRIS dye Consequently under denaturing conditions ven i the two strande migrate at diferant rats oniy the one labeled strand is detected Because of this you can vol spit peaks which result when two Stands move though a denaturing gel at dierent rates Under denaturing condions you can achieve a linear range of separation for fragment sizas of up to 500 bases Figure 7 on paga 32 measure ot how wel the standard defniton matches the GeneScan size standard and whether or not tis near To algn the data by size GoneScan calculates a best least squares curve for all samples This isa third order cure when you use the Third Order Least Squares size cang method For al othar size Celing methods tis a second order cure
23. lification EI amain Ve wig a sas OL REGTIG nba 10011 preselectie srmpittcation reaction product 190044 TEn butler Tarot en sin Gan a Tage kr T0 sanda Ek use inmate Ampl he EcoRI and Meol modifed fragments EI Sens Ve along EE bs BERE ea Geneamp PCR System 0800 ar 2400 DEL rhe DNA Thermal raso 0 hs reel aplican ection product tout MeelPrine Oodels phi 1OuLESAIDYE pimer Aol at yt 150pL AFLP Coro Max Net Mung ta ONA mal yer ao zo arr Pan POR ung ha banal EE TEE Show Tae A ren Note For the GaneAnp POR System 9800 and DNA Thermal ele 430 tera ramp imes ae 0 0 second For the onsAnp PCR System 2400 ere al ramp ness 90 EE Tabie 4 Thermal cycler parameters for selective ampiication kou e sc a pr e ese ee ae ee ere E save save sre sav ze ne ne ne ne ES ES ES ES ES ES Zo Evaluating Results Overview Run Modales Preparing the Loading Buffer for the ABLS73 and ABI PRIS 377 You can evaluate the raul of the AFLP reactions by using GaneScan software to analyz data from samples loaded and run on the ABI 373 or ABI Prisu 377 DNA Sequencer or on the ABI PR 310 Genetic Analyzer Tho following instructions describe sep by sep procedures for lading samples and perorming electrophoresis on iese instruments Tho ABI 373 DNA Sequencer uses Fiter Set A The ABI Pris 377
24. ly and Amplification predominate nthe final product Note Keop al reagents and tubes on ize unloaded thermal cyl EI Seine Ve along WE POR nin be GEREED Geneamp PCR System 9800 ar 2400 0 5m or he DNA Thermal SE 40 ul Ged DNA prepared by anale gation TUE AFL prestlectve primar paire TED AFLP Coro Max Note it using the DNA Theme Oyeler 480 overtay your alee v 20 Ho hg minera ol TIGE EE IG Ag Fan ba loving POR mated ee aap tee an GOT 1 secon cn ma GensAmp PCR System 960 and DNA Trama Celar 40 090 onthe Gane PER System 2400 Boe BEE aha ania Table Thermal cycler parameters for praselective eigenen nou GES noo now ELI Ee wo ge pwe ge re ze aosee 2min 30min wee Verifying Run an agarose gol to see that ampliiaton has occurred Successful Amplification se RO O TG IE Sian te gawi nm bani WARNING Ethidium bromide lea powet mutagen and is moderately toi Wes loves a la cost and sataty asses when using a dye Van ra goon a UV mananan Aaa ot prea fom 00 1500 bp shouid be deat vile gue 6 ra hat lamas pl ls e rg eg SS l ena 1pgotDNA bi Zen TR EN Figuras Gel tous ater resbcton digestion ot 1 3 ug of DNA at and ahe prese amaron nh Preparing Prepare the preselective amplification products for selective Template ampiteaon Selective Amp
25. menga Fax Bets Mat EE Ta messe Fax 44 011025 252502 EI ek Ata 7007777 Fax 410417800878 Sout Eas Europe agra Cata ada Essen Coes and War Aca onza hal ek mum en Fax a0 0139 8989 a09 ca Engh Speaking ana West Asa Fanande ou Aes AT Oe Couns Nek ae gen UR Tat mmm Fax 44 011925 282500 Japan apan IRA PRE TORO Faster Ala Cha Oceania EE Em OO alicia RE What You Will Need to Perform AFLP Overview You wil ned the folowing 4 DNA_trom0 06 0 ug of good quality DNA depending onthe genome size The plant mapping kis are optimized for smal Genomes of 80 500 Mb and medium regular genomes of 500 6000 Me 4 AFLP KA Mods and materials as specified on pages 7 8 16 19 andin Appendix E Related Consumables and Accessorie page 50 AFLP Kit Modules The organization of the AFLP Plant Mapping Ki no individual modules aowe for maximum febli You can purchase individual modules Separately depending on your research goals as shown in Tatile 2 Table 2 What to Order Sanger Teaser Genomes Genomes Module 600 2000 mb Lesen Tipaton and Preseectvs FR 402004 Praza Ampitcaton Aplikasion Core Mix PNE Pa Selective Amplteaion PN 503050 PIN OST StartUp a an Ina primer nual paner paka oo Moa and Par ane sland Gro Econ Matyou re ER atou eaten ton seo ableton pagsara pagea 7 8
26. n Primers from Table 1 Table 1 AFLP Selective Ampliicaton Primers EcoRI Primer Regular Plant Genomes Primer 250reactons 500 reactions EAT NG CG ESSRLACAFAM 402088 pw emt ueren om KS mac 4902055 pawa EPORIAGONED om aa EMRIMGJOE wne wm ema ze popa PS maner ween pd EsoRi Primare Small Plant Genomes Primer 250 reactions Coen aa ERRETE FAM pd EzoRi AG FAM wem eneen 402955 00 rectora SES wee ema JOE pd He we Testing New Genomes Fluorescent Dye abeling and Marker Detection Table 1 AFLP Selective Amplification Primers ented Meal Primera Regula and Smal Pani Genomes Primer 250 reactions 500 reactions WC wes Meek ES Meck pans Meet RT ES Wegen ES De Sa Wong ES Wa pen M other genomes are to be tested you need to be sure that they reset appropriately wih these enzymes In general the Regular Plant Genome Ka should produce A genetic fingerprints wih genomes af 10210 6x 107base par and the Smal Plant Genome Ki wah genomes of 5x 107105 x10 basa para Emplticalgidelnos suggest hat ifthe G C content of the genome is 565 Mee wil not ghe a significant number fragments Optimal resaltar obtained wi Meel when the G C e EcoRI lso tends to produce more fragments in G C poor genomes In cases bere an organism s G C content Er restcon enzymes must ba determined empifcaly Appied Biosystems has adapted the AFLP technique for use wih our ABI Pou fuorescont dye labaling and d
27. nomes 500 6000 Mb PIN 402004 Smal Plant Genomes 50 500 MB PIN 402273 Tris module contains the folowing fve tubes 4 Adaptor pairs that allow you to perio the igation reactions during preparation of your genomic DNA template one tube of EcoRI adaptor paire one tube of Meel adaptor paire 4 Preseleciva primers one tube Preselecivo Amplification mix butler dNTPs MgCl and enzyme necossary to perform tha Preselectve PCR ampifcaton reactions one tbe 4 AFLP Referenca DNA you can us for a control one tube Suficient reagents are supplied to perform up to 100 of each of these reactions Soo Preparing Enzyme Master MB on page 21 forthe roageris needed for igation and praselective ampiieation Amplification Core Mix Module AFLP Selective Amplification Start Up Module Storage and Stability of Kit Components Tho AFLP Amplification Core Mix Module contains all ofthe components necessary to ampily modified target sequences This module contains fe tubes of Core Mix containing buler nucieotdes and AmplTage DNA polymerase Tho Gore Mix Module contains sufficient reagents for 500 amplification reactions of target genomic sequences You determine how the Selection occurs by choosing primer pairs rom the AFLP Selective Anpifleston Star Up Modul or pairs of individually sod primers To screen primer combinations use the AFLP Selective Amplification StartUp Module Regular Plan Genomes PIN 4303050
28. t ONA alan AFLP Amplification getan and Presdecive Ampicaten Mode PIN 402004 kr Regular Plant nomen and 40223 or Smal Plant Genomes hs purpose To prepare samples tor the AFLP Precolactve Amplicacn and AFLP Selective Amplification reactions you must meal the adaptor pairs 4 prepare a restriction igation enzyme master mix 4 prepare the restriction gato reactions diie the restitonigation reactions Annealing You must anneal the adaptor pairs supplied with the AFLP Ligation and Adaptor Pairs Master Mix Preselctve Amplieaton med bear you can use them for ina restieilon gallon reactions E From te AFLP patna resis enee vee Be boer bid al Adaptor Par anda Ape 3 low tubes to coat mom tempera ovr a Tomita red REEL Prepare an Enzyme Master Mic to poto the restricion igaton reactions for all 100 DNA samples or a proportonate amount for fewer reactions E RE 10 LL 10X TA DNA ipase butter wh ATP NEO DEEN 100 Wel Units DNA Ligase or 670 cohesive and igation unta IMPORTANT Uso high concentration preparations of tne enzymes o soid exceeding 5 gen me resans AG sae tod too bg he al vo TOL i SAN Goan Ts SSIS TO STA Sree ETER qT PANG ROAR BE IMPORTANT Fr bes rasute use the Enzyme Master Mix abi 1 2 our Do not store the Enzyme Mater Mex beyond te Gay on which tis bo sed DETA DAA Lan ar wit ATP Sn o NG 171 10 RANGE TOA EN Prepar
29. ting AFLP procedures cone Sage GE TE ETER Resin eene epost o mortar Tani conoi DNA NE corect temperature contral pogam parameters retro o Gens Pan System 6800 Users Mansa Gavan POR Syste S600 Agn 900 Va via spa afer misaligned ko twang 1e top porton coke For DNA Mama yak 280 Fater to he DNA Therma eessen yet 480 Users Manual peng amor Celta pipa tach pa ry Fr EL Piacoa reapers in apex ot ube totem ar ube Sanity aner combining Combined reagent eran Pubes n bok mea aer lampara or on ce bor awarded reagents ar combined Free tine Bia pesto viso Catamiaton wih agus Use aprons RAT When sampleisiocwn DNA wo vod orig DNA beman BNA toma ising laboratory nanding singe source Teang resale ar Igan vat te DNA again and epea EER sampes nadama nare Waa aa Ba sampa IN Song inte asosampier t95 C for mos pri lasang ng Ed Es erc Eco KE a n evan POF alg ans DNA loa less sample duo ES Table 18 Toubleshooting AFLP procedures ens ET Poesie Causes EE inal conna pats Oaea or mishandled angen Check pation dano E reagent Sir and use according Compare wh ech reagents ET Bie used primer a 25 C Donat expos resan deal piman to gt rong paroda ot ume TRENT aang ES row DNA sample parameters nays parameters Change arecalng mana Use samo size cen mead Eer PET sde ET EIE Chea Log arme reso a
30. tion fragments Preparation and by using two restriction endonucleases EcoRI and Mea Double Adaptor Ligation Se adaptors suppled wih each kit ar ligated to tho ends of the DNA fragmeris generating template DNA or subsequent polymerase chain racton POR wb Position and ligation taka place in a single reaction Ligation of the adaploroigonuclslide to the restricted DNA doesnot regenerate the reeognton site so rastriction does not recur after igaton Figure 3 A Out gram oA o a Mou ant eo AmA B Upata actor Cen NEE and Msoi lady genomic ONA sgn We Meo Een Gen La Tm Dee Template preparaton and Igan of AFLP adaptors Presclective The sequences of the adaptors and the restriction site serve as primer Amplification binding sies for a subsequent low level selection or preselectie mplicaton ofthe restriction fragments Tho Meel complementary primer contains a 3 C The EcoRI complementary primer contains a 3A Regular Pant Genome KA modules or no base addon Smal Plant Genome Ki modul Only those genomic fragments that have an adaptor on each end amply exponential during PCR ampiicaton Figure 4 Thie step flecvely purifies the target away from sequences that amaliy only Tear La those with one modifed end Preparet Tempe Gana DUA Famer oid wi sapere 7 DEE E Am Pipo Paci anton oe pape ong Selective Addtional PCR amplifcations ae run t further reduce the complet Amplification oft
31. upport at 1 800 831 6844 and select he apropo option bao tor suporton the product of your choice at any time rng the ca To Se Ee SE Analyzer la id EI See gaas For Suppor On Tis Sequencing ye Tou ieee Sg Na eier Te wespe Jee Sa Ses e e FoR and Sequence Ser FR SEU ZE TOU Telephone TAX Zeene ni E EE s WK El 6505355961 aa a EE n De aa TS met men For Sapper On Tie TONG Telephone Documents on Free 24 hour access to Applied Biosystems technical documents Demand including MSDSs i available by fx or e mail You can access Documents on Demand through the internet or by telephone Nova eae BS aag You can sar fr documents or ving keyword Up othe document can be sed read to you S TALE ELE waa mana ae Zoe your ax number ready Canada n Presto order an index of avalatie documents and rave kato you Each document ina dex has an 10 abe Ue sa our order number in stop ben a Cal 1 800 487 8800 trom auctions phone a 4 Press 20 order up to tve documents and have them edo you Ey phone tam a Di your eel saree code han ne REEN Lied Sates or Ho your complete te uber and country code Canada ready 01
32. y Manual Ses Appendix D on page 48 for specife references Appendix D References Ausubel FM Brent R Kingsiin RE Moore DI Seidman 16 Smith JA and Sirini K ede 1987 Current Protocole in Molecular Biology Greene Publishing Associates and Wi ntersderee John Woy and Sons New Yon Baker S B Rugh CLL and Kamalay LC 1990 RNA and DNA isolation ram recalcitrant plant tissue Biotechniques 268 272 Batas SALE Knorr DA Weler JN and Ziego JS 1996 Instrumentation for automated molecular marker aquisiton and data analysis In Sobral BW 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 Mol Gen Genet 240 65 73 Doyle J and Doyle J 1990 elation of plant DNA rom tresh tissue Focus YE 13 46 Maksem K Leister D Peleman J Zabeau M Salamini F and Seen 1998 A high resolution map of ihe At locus on chromosome V of potato based on RFLP and AFLP markers Mol Gon Genet 249 74 81 Sambrook J Fritsch EF and Maniatis T 1989 Molecular Cloning A Laboratory Manual Gold Spring Harbor Press NY Thomas CM Vos P Zaboau M Jones DA Norco KA Chadwick B and Jones LD 1995 Identification of ampled restriction agent polymorphism AFLP marters tightly inked to the tomato CL gene for resistance to Cladosporum fuum Plant J SSC Van
33. y units Al sample fragments were sized using the GoneScan 500 ROX siza Standard Elocropherogram data and tabular data were generated Using GenaScan Analysts software version 20 Figure Expanded eacropherpram and size data for AFLP sampi Evaluating An elacropherogram of E col W3110 Reference DNA run on an ABI PRISM 310 ABI Pris 310 Genetic Analyzer is shown in Figure 12 The Mei CA Genetic Analyzer and FAM labeled EcORI A selectivo primer from the AFLP Microbial Results Fingerprinting K P N 402048 were used Note Ters are kg iarncas in agent sizes on tw ABI Pr 10 compared oth ABI 373 and ABI Pre 377 L Figure 12 ABI Pris 310 electophargram of Ecol W3110 Reference DNA Appendix A Primer Combination Tables Genomes Analyzed Ten diferent crop species genomes wore analyzed using the AFLP Using AFLP technique For each crop species primer combinations that produce the best DNA fingerprints wore determined Tho names of each cop species testad and corresponding primer combination taos are given in Table 7 Those combinations of EcoRI and Mao Selective Ampliicaton primers that ar best suited for amplfcaon screening ofthe designated crop genomes are shown in able 8 through Table 17 Table Primer combination tables for crop species Crop Species Primer Combination RE Regular Pant casas Suntoner able 8 on page 39 Pepper Tate 9 on page 39 Batley abe 1
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