SSR技术原理,方法及步骤课件

1、SS叱术1. SSR简介说明：简单重复序列(SimpleSequenceRepeat,SSR)简单重复序(SSR)也称微卫星DNA,其串联重复的核心序列为1-6bp,其中最常见是双核甘酸重复，即(CA)n和(TG)n每个微卫星DNA的核心序列结构相同，重复单位数目10-60个，其高度多态性主要来源于串联数目的不同。SSR标记的基本原理：根据微卫星序列两端互补序列设计引物，通过PCR反应扩增微卫星片段，由于核心序列串联重复数目不同，因而能够用PCR的方法扩增出不同长度的PCR产物，将扩增产物进行凝胶电泳，根据分离片段的大小决定基因型弁计算等位基因频率。在真核生物中，存在许多2-5bp简单重复序列

2、，称为“微卫星DNA具两端的序列高度保守，可设计双引物进行PCR扩增，揭示其多态性。SSR具有以下一些优点：(l)一般检测到的是一个单一的多等位基因位点；(2)微卫星呈共显性遗传，故可鉴别杂合子和纯合子；(3)所需DNA量少。显然，在采用SSR技术分析微卫星DNA多态性时必须知道重复序列两端的DNA序列的信息。如不能直接从DNA数据库查寻则首先必须对其进行测序。SSR的分类：根据SSR核心序列排列方式的不同，可分为3种类型：1)完全型(perfect),指核心序列以不间断的重复方式首尾相连构成的DNAo如：ATATATATATATATATATATATATATATATATAT;2)不完全型(im

3、perfect),指在SSR的核心序列之间有3个以下的非重复碱基，但两端的连续重复核心序列重复数大于3。如：ATATATATGGATATATATATCGATATATATATATATATGGATATATATAT;3)复合型(compound),指2个或2个以上的串联核心序列由3个或3个以上的连续的非重复碱基分隔开，但这种连续性的核心序列重复数不少于5。如：ATATATATATATATGGGATATATATATATA。3种类型中完全型是SSR标记中应用较多的一种类型。SSR在植物基因组中的分布:SSR广泛分布于各种真核生物的基因组中，大约每隔1050kb就存在一个SSR哺乳动物中的SSR的数量大约

4、为植物中的56倍。在植物中，平均23.3kb就有一个SSR双子叶植物中的SSR数量大于单子叶植物，前者两个SSR之间的平均间距为21.2kb,后者为64.6kb;核DNA中的SSR数量多于细胞质DNA中的SSR绝大多数单碱基重复型及2碱基重复型SSR存在于非编码区，3碱基重复型多位于编码区。微卫星的利用价值:由于核心序列重复数的不同，等位的SSR位点可呈现出多态性（SSLP,simplesequencelengthpolymorphism）。大多表现为共显性遗传，有的表现为显性遗传。由于SSRDNA两侧序列（离开20bp以上）表现出保守特征，所以可设计出上下游PCR引物，扩增出包含SSR的DN

5、A序列。微卫星分析常用于：遗传图谱构建；种质鉴定；遗传多样性分析；标记辅助选择（MAS,marker-assistantseletion,marker-aidedseletion）；基因定位；数量性状基因座（QTL）分析；系谱分析；亲源关系鉴定等。SSR引物的来源：借鉴其他近缘种序列。1）通过筛选文库、测序开发自己的SSR引物。2）通过核酸数据库查询，从已有序列中搜寻包括SSR的序列弁设计引物。SSR分析实验的主要技术环节：提取DNAPCR扩增；电泳及显色；电泳胶板带型的照相、记录；数据分析处理。其中，PCR产物分离的电泳方法主要有：高浓度琼脂糖电泳（4%交只能分辨4-6bp差异）；变性聚丙烯

6、酰胺序列胶电泳；非变性聚丙烯酰胺凝胶电泳。由于扩增的片段短（一般小于300bp）,基因间的差异小（一般为几个bp）,故通常使用分辨率高的聚丙烯酰胺凝胶电泳。在程序上，变性胶虽然比非变性胶麻烦些，但考虑到在非变性胶上会出现人为假象一异源双链分子，比如导致SSR杂合子中出现3-4条带，而不是正常的2条带，从而干扰等位位点统计，因此我们建议在SSR分析中均采用变性胶电泳。2. ISSR分子标记的实验原理及操作流程原理：ISSR（inter-simplesequencerepeat）标记是一种类似RAPD但利用包含重复序列弁在3'或5'锚定的单寡聚核酸引物对基因组进行扩增的标记系统，即

7、用SSR引物来扩增重复序列之间的区域。其原理具体是，ISSR标记根据之物广泛存在SSR的特点，利用在生_物基因组常出现的SSR本身设计引物，无需预先克隆和测序。用于扩增的引物一般为16-18个碱基序列，由1-4个碱基组成的串联重复和几个非重复的锚定碱基组成，从而保证了引物与基因组DNA中SSR的5'或3'末端结合，导致位于反向排列、间隔不太大的重复序列间的基因组节段讲行PCR扩增。一、实验材料不同来源的DNA（30-50ng/ul）。二、实验设备PCR仪，PCR管或硅化的0.5mleppendorf管，电泳装置，凝胶成像仪。三、试剂1、ISSR引物：购买成品或根据加拿大Brit

8、ishColumbia大学设计的ISSR引物序列（见附录）自己合成2、Taq酶3、10xPCR缓冲液4、MgCl2：25mmol/L5、dNTP:2.5mmol/L。四、操作步骤：1.在25ul反应体系中，加入2.模板DNA1ul(30-50ng)10xPCRBuffer2.5uldNTP2ul加ddH2O至25ul在PCRt中预变性ISSR引物1ul(约5pmol)MgC22ulTaq酶1单位（U）混匀稍离心加一滴（约20ul）矿物油94C2分钟，然后循环：94C1分钟，45C-68C40秒,72C1-2分钟，共40轮循环。3.循环结束后,72C10分钟，4c保存。4 .取PCR产物15ul

9、加3ul上样缓冲液50-100V（电压低带型整齐，分辨率高）。5 .电泳结束，澳化乙锭染色20分钟。6 .用凝胶成像仪观察、拍照。操作流程简图：（6x）于1.6%或1.8%琼脂糖胶上电泳,稳压wax*3.SSRGELandSilverStainingProtocolPaulaMarquardt&CraigEchtPublishedin:EchtCS,May-MarquardtP,HseihM,ZahorchakR.1996.Characterizationofmicrosatellitemarkersineasternwhitepine.Genome39:1102-1108.Comme

10、ntscanbedirectedtoPaulaMarquardtat:USDAForestServiceResearch5985HighwayKRhinelander,WI54501USAPhone:715-362-1121Fax:715-362-1166e-mail:pmarquar1 .EQUIPMENT:DNAsequencingunit(35x45cm)&2000VpowersupplyClampsLg.plastictrays(4),about43x50x8cm,andonelidTworockingplatformsHeatblockformicrotiterplates.

11、Amicroplatevortexerishelpful.II.MOLDASSEMBLY:Notes:Bindsilaneistoxicandshouldbeusedinachemicalfumehood.Weargloveswhenhandlingthissolution.Useasmallpieceofvinyltapeonaloweroutsidecorneroftheacryleasetreatedglassgelplatetomarktheuntreatedsideandalsohelpdistinguishtheplates.Thishelpsavoidconfusionbetwe

12、enplateswhenusingoffsetplates.Thetapecanremaininplacethroughseveralelectrophoresis/washingcycles.1. Washinnerandouterplateswellwithalconoxcleanser.Rinsewellwithtapwater,deionizedordistilledwater,andethanol,airdry.Usededicatedspongesforeachtreatment.2. Usingakimwipetissue,coattheinnersideofnotchedoro

13、ffsetplatewithacrylease(Stratagene)andallowtodry-Idonottreatthetop2inchesoftheplatesinceIfeelthatthenonstickcoatingpromotesleakingbetweenwellsbehindtheteethofthecomb.Buffwellwithakimwipesoakedinethanolforacleanfinish;thistakessomeelbowgreasetogetthestreaksoffoftheplate.Changeglovesbeforeworkingwithb

14、indsilaneandtakecarenottocrosscontaminatetheplateswiththetwotreatments.Theacryleasetreatmentonlyneedstoberepeatedeveryfourgelsorso.3. Preparefresh1mlbindingsolutionbymakingasolution0.0005-0.001%bindsilane(Sigma#M-6514)in95%ethanol,0.5%glacialaceticacid.Applywithakimwipeandcoattheinnersideofthelarger

15、platewithonemlandallowtodry4-5minutes.Wipetheplatewithethanolinonedirectionandthenperpendiculartofirstdirection,don"tusetoomuchpressure.Thistreatmentneedstoberepeatedeverytime.III. GELSOLUTIONPREPARATION:Note:Acrylamideistoxic.Weargloveswhenhandlingsolutionandfacemaskwhenweighingoutpowder.Asafe

16、ralternativeistobuyapremix.1. Rinseallglasswareandplasticwarewithd.i.waterpriortogelsolutionpreparationandpouring,includingthedisposablefilterunit.2. Gelsare6%acrylamide,8Murea,1XTBE.Foreachgel,mixtogether50gurea,15ml40%19:1acrylamidesolution,and31mld.i.water.Weusea4.5%gelforfingerprintingreactions.

17、Note:(Westorealiquotsofpremixed40%Acrylogelsolution,Gallard-SchleisingerInd.,at-20C.)3. Warmandstirthemixtureinabeakerofwarmwateruntilalltheureaisdissolved.Add1.25gofamberliteresinandstir5min.Filterthrougha0.2uMfilteranddegasat25mgHgfor5min.Transfertograduatecylinderandadd10ml10xTBE,bringingvolumeto

18、100mlwithd.i.water.4. WehaverecentlystartedusingBurst-PackfromOwlScientific,whichisanacrylamidepremixincludingthebufferandcatalysts,forourfluorescentgelworkandareverypleasedwiththequalityandreproducibility.Thiswouldbeanoptionforthesilverstainingworkaswell.Theburst-pack"seliminatethechemicalweig

19、hingandmixing,deionizing,filteringanddegassingsteps.IV. GELPOURING:1. Immediatelypriortopouring,add500ul10%ammoniumpersulfate(0.1g+1mld.i.water)totheacrylamidemixinabeaker,gentlymixingwell.Thenadd50ulTEMEDandmix.PolymerizationwillnotstartuntilTEMEDhasbeenadded.Donotmixthecatalyststogetherbeforeaddin

20、gtothepolyacrylamidesolution-thiswillinhibitpolymerization.2. WeusetheOtteradjustablegelcaster(OWLScientific,Inc.)forpouringgels.Inthissystemthegelispouredhorizontallywiththetopplateslidingoverthebottomplate,withouttheuseoftape,greaseorabottomspacer.a. Placethelargerplateontocastersothatitabutstheen

21、dwallofthecaster.Moistenspacerswithwaterandplacethemflushtotheedgeofglassagainstthecasterwall.b. Placethetopplate(notchedoroffset)sothatitstopedgeoverlapsthebottomedgeofthelowerplateby3-4cm.Usinga60mlsyringe,slowlydispensethegelsolutionbetweentheplates,allowingthesolutiontoflowbycapillaryaction.Gent

22、lyslidethetopplateacrossthebottomplatewhiledispensingthegelsolutionalongtheleadingedgeofthetopplate.Ifanybubblesformwhilepouring,tryslidingthetopplatebacktouncoverthebubble,thenproceed.Amoreeffectivemethodistodragoutthebubbleswithaplastichook(freebyrequestfromPromegaCorp.)3. Oncethegelispoured,inser

23、ttheflatedgeofasharktoothcomb(oracastingcomb)intothetopofthegeltothedepthdesiredforthewells.Place2-3clampsalongthesidesandtoptokeepplatesintightcontactwiththespacersandcombwhilethegelispolymerizing.4. Allowthegeltopolymerizeatroomtemperature(RT)for1hour.GelcanbestoredatRTovernightifstepsaretakentopr

24、eventitfromdryingout.Todothis,placepapertowelsdampenedwithrunningbufferoverthetop(removeclampsbutleavecombinplace)andbottomedgesofthegelmoldandwrapwithplasticwrap.Donotstorethegelunderbuffer.V. SAMPLEPREPARATION:Notes:Heatsamplesimmediatelypriortoloading.Keeptheloadingdyefresh.UseSSRPloadingdyethati

25、slessthan2weeksold.Thedeionizedformamideusedinmakingtheloadingdyeshouldbelessthanonemonthold.1. DenaturethesampleDNAbyadding1volume(10ul)offreshSSRPloadingdye(10mMNaOH,95%formamide,0.05%bromophenolblue,0.05%xylenecyanol)to1volumeofPCRsampleinamicrotiterplate.Mixwellandheatto95oCfor2min.Placeonice.2.

26、MolecularweightstandardsarePGEM(Promega)andPoly-dA(Pharmacia#27-7836-01)sonicatedtoproducea1bpladder.PGEMisloadedinawellseparatefrompolyA.WedonotusepolyAforfingerprintinggels.Usinga144-well,microtiter4Xoffsetcomb,load3ulofthemix:3.4ul1XPerkinElmerIIPCRbuffer8.6ulof30ng/ulPGEM12ulofSSRPbufferheat95Cf

27、or2min.,iceand5ul1XPEIIbuffer2.5ulof400ng/ulofsonicatedPoly-dA7.5ulSSRPbufferVI .ELECTROPHORESIS:Note:Addingsodiumacetatetothebottomreservoirduringelectrophoresis(SheenandSeed,1988,Biotechniques6:942-944)producesthesameeffectasrunningwedgeshapedgelsoraddingagradienttothegelsthemselves(Bigginet.al.,1

28、983,PNAS80:39633965).Inallthreemethods,themobilityofsmallDNAfragmentsisrestardedastheyapproachthebottomofthegel.Thesodiumacetatemethodissimplerthantheothermethods.1. Removeclamps.Cleanexcesspolyacrylamideandureafromthetopofplateassemblywithd.i.water.Pullthecomboutofthemoldslowlyandevenly,cleaningout

29、thecombareawithd.i.waterorbuffer.2. Addreservoirbufferstotheapparatus.Thetopreservoirbufferis1XTBE.Thebottombufferreservoiris2/3XTBE,1Msodiumacetate.Make1500mlbottombufferforeachgel(100ml10xTBE,900mld.i.water,500ml3MNaAcetate).3. Pre-electrophoresefor5-10mintowarmtheplatesothatthecombwilleasilyslide

30、inplace.Cleanoutcombareawithbuffer.Topreventpossiblewelltowellleakage,applyaverylightcoatingofcellosealtotheoutsidesurfaceofthecomb,priortoinsertion.Placeplateassemblyingelboxandclamp.4. Formicrotiterformat,ahamilton8or12channelsyringeloadingdeviceforloadingthegelsisrecommended.Cleanouteachgroupofwe

31、llsimmediatelypriortoloadingwiththemultichannelhamiltonsyringe.Runthegelat50Cconstanttemperatureand100wattslimitingpowerforabout1.5-3hours,dependingonsizeofamplificationproduct.ConstanttemperaturecanbemaintainedwiththetemperatureprobeoptionoftheBioRadPower/Pak3000powersupply.VII GELFIXING,STAININGAN

32、DCOLORDEVELOPMENT:Notesandtips:-ThisprocedureisadaptedfromthePromegaSilverSequenceprotocol.-Usehighestqualityreagents.-Thefixer,stainanddeveloperaremadewith18mega-ohmwater(deionized=d.i.).-Thewashesaredonewithdistilledwater.-Weusepre-measuredsodiumthiosulfate(-20C)andsilvernitrate(RT),storedinmicrot

33、ubes.-AllincubationsandwashesareCaswell,fortheperformedatroomtemperature,butthedevelopersolutionmustbepre-chilledto4-10Ctominimizebrownbackground.-Itisimportanttokeepthestopsolutionat4-10samereason.-Solutionscontainingformaldehydeshouldbehandledinafumehood.Theformaldehydeisaliquoted(RT)-Wearglovesth

34、roughouttheprocedure.-Wearinganapronwillpreventsilverstainsonclothing.-Rockingplatformsarebetterthanorbitalshakerstoachieveevengelstaining.1. Freshlypreparestaininganddevelopersolutionswhilegelisrunning.Addformaldehyde,silvernitrateandsodiumthiosulfatetosolutionsimmediatelypriortouse.Topreparestaini

35、ngsolution,combine2Ld.i.water,2gsilvernitrateand3ml37%formaldehyde.Topreparedevelopingsolution,combine2Lchilledd.i.water,60gsodiumcarbonate,3ml37%formaldehydeand4mgsodiumthiosulfate.Chilldeveloperto4-10C.2. Removegelassemblefromrig,removesidespacersandcarefullyseparateglassplateswithaspatula.Thegelw

36、illremainattachedtotheplatecoatedwithbindsilane.Cutoffthetopcornerofthehighnumberedendofgelfororientation.3. Placegelandplateinashallowplastictraycontainingsufficientfreshfix/stopsolution(2liter7.5%glacialaceticacid)andgentlyagitatefor20minutesoruntiltrackingdyecompletelydisappears.Wesometimesuseamu

37、ltiplegelholderfromPromegaforprocessingseveralgelsatatime.Twogelsrequirethesamevolumeofsolutionasone.Forthreegels,solutionvolumesareincreasedby50%.Note:Forovernightstorage,thegelcanbefixedasabove,rinsedwithd.i.waterandstoredinfreshfixerwithoutagitation.4. Rinsethegel3timesfor2minuteseachwithdistille

38、dwaterwithgentleagitation.5. Addstainingsolutiontogelandgentlyagitatefor30minutes,decantandprecipitatesilver.ThesilverintheusedstainingsolutionisprecipitatedwithNaCl(10g/2L)forrecycling.6. Rinsegelwithdistilledwaterfor5seconds(fromtimegelisplacedindistilledwatertotimeintodeveloper,i.e.aquickdip).Lon

39、gerrinsesresultinweakersignal.Iftherinsegoestoolong,repeatstep5withthestainingsolution.7. Transferthegelto800mlsofchilleddevelopingsolutionandgentlyagitatewitharockingmotionuntilthebandsfirstbecomevisible,usuallywithininafewminutes.Decanttheuseddeveloper,addtheremainingchilleddeveloperandcontinuetogentlyagitateuntilthebandshavereacheddesiredintensity.Over-developmentwillresultinabrownbackgroundwithlowcontrastbandsratherthanapencilgraybackgroundwithsharpbands.8.