1、SSR技術(shù)1. SSR簡介說明： 簡單重復(fù)序列(SimpleSequenceRepeat,SSR),簡單重復(fù)序(SSR)也稱微衛(wèi)星DNA,其串聯(lián)重復(fù)的核心序列為1-6bp,其中最常見是雙核甘酸重復(fù)，即(CA)n和(TG)n每個(gè)微衛(wèi)星DNA的核心序列結(jié)構(gòu)相同，重復(fù)單位數(shù)目10-60個(gè)，其高度多態(tài)性主要來源于串聯(lián)數(shù)目的不同。SS刖記的基本原理：根據(jù)微衛(wèi)星序列兩端互補(bǔ)序列設(shè)計(jì)引物，通過PCR反應(yīng)擴(kuò)增微衛(wèi)星片段，由于核心序列串聯(lián)重復(fù)數(shù)目不同，因而能夠用PCR的方法擴(kuò)增出不同長度的PCR產(chǎn)物，將擴(kuò)增產(chǎn)物進(jìn)行凝膠電泳，根據(jù)分離片段的大小決定基因型并計(jì)算等位基因頻率。在真核生物中，存在許多2-5bp簡單重復(fù)

2、序列，稱為微衛(wèi)星DNA其兩端的序列高度保守，可設(shè)計(jì)雙引物進(jìn)行PCR擴(kuò)增，揭示其多態(tài)性。SSR具有以下一些優(yōu)點(diǎn):(l)一般檢測到的是一個(gè)單一的多等位基因位點(diǎn)；(2)微衛(wèi)星呈共顯性遺傳，故可鑒別雜合子和純合子；(3)所需DNA量少。顯然，在采用SSR技術(shù)分析微衛(wèi)星DNA多態(tài)性時(shí)必須知道重復(fù)序列兩端的DNA序列的信息。如不能直接從DNA數(shù)據(jù)庫查尋則首先必須對其進(jìn)行測序。SSR的分類:根據(jù)SSR核心序列排列方式的不同，可分為3種類型：1)完全型(perfect),指核心序列以不間斷的重復(fù)方式首尾相連構(gòu)成的DNA。如：ATATATATATATATATATATATATATATATATAT;2)不完全型(i

3、mperfect),指在SSR的核心序列之間有3個(gè)以下的非重復(fù)堿基，但兩端的連續(xù)重復(fù)核心序列重復(fù)數(shù)大于3。如：ATATATATGGATATATATATCGATATATATATATATATGGATATATATAT;3)復(fù)合型(compound),指2個(gè)或2個(gè)以上的串聯(lián)核心序列由3個(gè)或3個(gè)以上的連續(xù)的非重復(fù)堿基分隔開，但這種連續(xù)性的核心序列重復(fù)數(shù)不少于5。如：ATATATATATATATGGGATATATATATATA。3種類型中完全型是SSR標(biāo)記中應(yīng)用較多的一種類型。SSR在植物基因組中的分布：SSR廣泛分布于各種真核生物的基因組中，大約每隔1050kb就存在一個(gè)SSR。哺乳動物中的SSR的數(shù)量

4、大約為植物中的56倍。在植物中，平均23.3kb就有一個(gè)SSR;雙子葉植物中的SSR數(shù)量大于單子葉植物， 前者兩個(gè)SSR之間的平均間距為21.2kb,后者為64.6kb;核DNA中的SSR數(shù)量多于細(xì)胞質(zhì)DNA中的SSR,絕大多數(shù)單堿基重復(fù)型及2堿基重復(fù)型SSR存在于非編碼區(qū)，3堿基重復(fù)型多位于編碼區(qū)。微衛(wèi)星的利用價(jià)值：由于核心序列重復(fù)數(shù)的不同，等位的SSR位點(diǎn)可呈現(xiàn)出多態(tài)性（SSLP,simplesequencelengthpolymorphism）。大多表現(xiàn)為共顯性遺傳，有的表現(xiàn)為顯性遺傳。由于SSRDNA兩側(cè)序列（離開20bp以上）表現(xiàn)出保守特征，所以可設(shè)計(jì)出上下游PCR引物，擴(kuò)增出包含S

5、SR的DNA序列。微衛(wèi)星分析常用于：遺傳圖譜構(gòu)建；種質(zhì)鑒定；遺傳多樣性分析；標(biāo)記輔助選擇（MAS,marker-assistantseletion,marker-aidedseletion）；基因定位；數(shù)量性狀基因座（QTL）分析；系譜分析；親源關(guān)系鑒定等。SSR引物的來源：借鑒其他近緣種序列。1）通過篩選文庫、測序開發(fā)自己的SSR引物。2）通過核酸數(shù)據(jù)庫查詢，從已有序列中搜尋包括SSR的序列并設(shè)計(jì)引物。SSR分析實(shí)驗(yàn)的主要技術(shù)環(huán)節(jié)：提取DNA;PCR擴(kuò)增；電泳及顯色；電泳膠板帶型的照相、記錄；數(shù)據(jù)分析處理。其中，PCR產(chǎn)物分離的電泳方法主要有：高濃度瓊脂糖電泳（4%膠只能分辨4-6bp差異）

6、；變性聚丙烯酰胺序列膠電泳；非變性聚丙烯酰胺凝膠電泳。由于擴(kuò)增的片段短（一般小于300bp）,基因間的差異小（一般為幾個(gè)bp）,故通常使用分辨率高的聚丙烯酰胺凝膠電泳。在程序上，變性膠雖然比非變性膠麻煩些，但考慮到在非變性膠上會出現(xiàn)人為假象一異源雙鏈分子，比如導(dǎo)致SSR雜合子中出現(xiàn)3-4條帶，而不是正常的2條帶，從而干擾等位位點(diǎn)統(tǒng)計(jì)，因此我們建議在SSR分析中均采用變性膠電泳。2. ISSR分子標(biāo)記的實(shí)驗(yàn)原理及操作流程原理：ISSR（inter-simplesequencerepeat）標(biāo)記是一種類似RAPD,但禾U用包含重復(fù)序列并在3或5錨定的單寡聚核酸引物對基因組進(jìn)行擴(kuò)增的標(biāo)記系統(tǒng)，即用S

7、SR引物來擴(kuò)增重復(fù)序列之間的區(qū)域。其原理具體是，ISSR標(biāo)記根據(jù)生物廣泛存在SSR的特點(diǎn)，利用在口物基因組常出現(xiàn)的SSR本身設(shè)計(jì)引物，無需預(yù)先克隆和測序。用于擴(kuò)增的引物一般為16-18個(gè)堿基序列，由1-4個(gè)堿基組成的串聯(lián)重復(fù)和幾個(gè)非重復(fù)的錨定堿基組成，從而保證了引物與基因組DNA中SSR的5或3末端結(jié)合，導(dǎo)致位于反向排列、間隔不太大的重復(fù)序列間的基因組節(jié)段進(jìn)行PCR擴(kuò)增。一、實(shí)驗(yàn)材料不同來源的DNA（30-50ng/ul）。二、實(shí)驗(yàn)設(shè)備PCR儀，PCR管或硅化的0.5mleppendorf管，電泳裝置，凝膠成像儀。三、試劑1、ISSR引物：購買成品或根據(jù)加拿大BritishColumbia大學(xué)

8、設(shè)計(jì)的ISSR引物序列（見附錄）自己合成2、Taq酶3、10 xPCR緩沖液4、MgCl2：25mmol/L5、dNTP：2.5mmol/L。四、操作步驟：1 .在25ul反應(yīng)體系中，加入10 xPCRBuffer2.5ulMgCl22ulTaq酶1單位（U）2.在PCR儀中預(yù)變性94C2分鐘，然后循環(huán)：94C1分鐘,分鐘，共40輪循環(huán)。3.循環(huán)結(jié)束后，72C10分鐘，4c保存。4.取PCR產(chǎn)物15ul加3ul上樣緩沖液50-100V（電壓低帶型整齊，分辨率高）。5.電泳結(jié)束，澳化乙錠染色20分鐘。6.用凝膠成像儀觀察、拍照。操作流程簡圖：3、SSRGELandSilverStainingPr

9、otocolPaulaMarquardt&CraigEchtPublishedin:EchtCS,May-MarquardtP,HseihM,ZahorchakR.1996.Characterizationofmicrosatellitemarkersineasternwhitepine.Genome39:1102-1108.CommentscanbedirectedtoPaulaMarquardtat:USDAForestServiceResearch模板DNA1ul(30-50ng)ISSR弓I物1ul(約5pmol)dNTP2ul加ddH2O至25ul混勻稍離心，加一滴（約20u

10、l）礦物油。45C-68c40秒，72c1-25985HighwayKRhinelander,WI54501USAPhone:715-362-1121Fax:715-362-1166e-mail:I.EQUIPMENT:DNAsequencingunit(35x45cm)&2000VpowersupplyClampsLg.plastictrays(4),about43x50 x8cm,andonelidTworockingplatformsHeatblockformicrotiterplates.Amicroplatevortexerishelpful.II.MOLDASSEMBLY:

11、Notes:Bindsilaneistoxicandshouldbeusedinachemicalfumehood.Weargloveswhenhandlingthissolution.Useasmallpieceofvinyltapeonaloweroutsidecorneroftheacryleasetreatedglassgelplatetomarktheuntreatedsideandalsohelpdistinguishtheplates.Thishelpsavoidconfusionbetweenplateswhenusingoffsetplates.Thetapecanremai

12、ninplacethroughseveralelectrophoresis/washingcycles.1. Washinnerandouterplateswellwithalconoxcleanser.Rinsewellwithtapwater,deionizedordistilledwater,andethanol,airdry.Usededicatedspongesforeachtreatment.2. Usingakimwipetissue,coattheinnersideofnotchedoroffsetplatewithacrylease(Stratagene)andallowto

13、dry-Idonottreatthetop2inchesoftheplatesinceIfeelthatthenonstickcoatingpromotesleakingbetweenwellsbehindtheteethofthecomb.Buffwellwithakimwipesoakedinethanolforacleanfinish;thistakessomeelbowgreasetogetthestreaksoffoftheplate.Changeglovesbeforeworkingwithbindsilaneandtakecarenottocrosscontaminatethep

14、lateswiththetwotreatments.Theacryleasetreatmentonlyneedstoberepeatedeveryfourgelsorso.3. Preparefresh1mlbindingsolutionbymakingasolution0.0005-0.001%bindsilane(Sigma#M-6514)in95%ethanol,0.5%glacialaceticacid.Applywithakimwipeandcoattheinnersideofthelargerplatewithonemlandallowtodry4-5minutes.Wipethe

15、platewithethanolinonedirectionandthenperpendiculartofirstdirection,dontusetoomuchpressure.Thistreatmentneedstoberepeatedeverytime.III.GELSOLUTIONPREPARATION:Note:Acrylamideistoxic.Weargloveswhenhandlingsolutionandfacemaskwhenweighingoutpowder.Asaferalternativeistobuyapremix.1. Rinseallglasswareandpl

16、asticwarewithd.i.waterpriortogelsolutionpreparationandpouring,includingthedisposablefilterunit.2. Gelsare6%acrylamide,8Murea,1XTBE.Foreachgel,mixtogether50gurea,15ml40%19:1acrylamidesolution,and31mld.i.water.Weusea4.5%gelforfingerprintingreactions.Note:(Westorealiquotsofpremixed40%Acrylogelsolution,

17、Gallard-SchleisingerInd.,at-20C.)3. Warmandstirthemixtureinabeakerofwarmwateruntilalltheureaisdissolved.Add1.25gofamberliteresinandstir5min.Filterthrougha0.2uMfilteranddegasat25mgHgfor5min.Transfertograduatecylinderandadd10ml10 xTBE,bringingvolumeto100mlwithd.i.water.4. WehaverecentlystartedusingBur

18、st-PackfromOwlScientific,whichisanacrylamidepremixincludingthebufferandcatalysts,forourfluorescentgelworkandareverypleasedwiththequalityandreproducibility.Thiswouldbeanoptionforthesilverstainingworkaswell.Theburst-packseliminatethechemicalweighingandmixing,deionizing,filteringanddegassingsteps.IV. G

19、ELPOURING:1. Immediatelypriortopouring,add500ul10%ammoniumpersulfate(0.1g+1mld.i.water)totheacrylamidemixinabeaker,gentlymixingwell.Thenadd50ulTEMEDandmix.PolymerizationwillnotstartuntilTEMEDhasbeenadded.Donotmixthecatalyststogetherbeforeaddingtothepolyacrylamidesolution-thiswillinhibitpolymerizatio

20、n.2. WeusetheOtteradjustablegelcaster(OWLScientific,Inc.)forpouringgels.Inthissystemthegelispouredhorizontallywiththetopplateslidingoverthebottomplate,withouttheuseoftape,greaseorabottomspacer.a. Placethelargerplateontocastersothatitabutstheendwallofthecaster.Moistenspacerswithwaterandplacethemflush

21、totheedgeofglassagainstthecasterwall.b. Placethetopplate(notchedoroffset)sothatitstopedgeoverlapsthebottomedgeofthelowerplateby3-4cm.Usinga60mlsyringe,slowlydispensethegelsolutionbetweentheplates,allowingthesolutiontoflowbycapillaryaction.Gentlyslidethetopplateacrossthebottomplatewhiledispensingtheg

22、elsolutionalongtheleadingedgeofthetopplate.Ifanybubblesformwhilepouring,tryslidingthetopplatebacktouncoverthebubble,thenproceed.Amoreeffectivemethodistodragoutthebubbleswithaplastichook(freebyrequestfromPromegaCorp.)3. Oncethegelispoured,inserttheflatedgeofasharktoothcomb(oracastingcomb)intothetopof

23、thegeltothedepthdesiredforthewells.Place2-3clampsalongthesidesandtoptokeepplatesintightcontactwiththespacersandcombwhilethegelispolymerizing.4. Allowthegeltopolymerizeatroomtemperature(RT)for1hour.GelcanbestoredatRTovernightifstepsaretakentopreventitfromdryingout.Todothis,placepapertowelsdampenedwit

24、hrunningbufferoverthetop(removeclampsbutleavecombinplace)andbottomedgesofthegelmoldandwrapwithplasticwrap.Donotstorethegelunderbuffer.V.SAMPLEPREPARATION:Notes:Heatsamplesimmediatelypriortoloading.Keeptheloadingdyefresh.UseSSRPloadingdyethatislessthan2weeksold.Thedeionizedformamideusedinmakingtheloa

25、dingdyeshouldbelessthanonemonthold.1. DenaturethesampleDNAbyadding1volume(10ul)offreshSSRPloadingdye(10mMNaOH,95%formamide,0.05%bromophenolblue,0.05%xylenecyanol)to1volumeofPCRsampleinamicrotiterplate.Mixwellandheatto95oCfor2min.Placeonice.2.MolecularweightstandardsarePGEM(Promega)andPoly-dA(Pharmac

26、ia#27-7836-01)sonicatedtoproducea1bpladder.PGEMisloadedinawellseparatefrompolyA.WedonotusepolyAforfingerprintinggels.Usinga144-well,microtiter4Xoffsetcomb,load3ulofthemix:3.4ul1XPerkinElmerIIPCRbuffer8.6ulof30ng/ulPGEM12ulofSSRPbufferheat95cfor2min.,iceand5ul1XPEIIbuffer2.5ulof400ng/ulofsonicatedPol

27、y-dA7.5ulSSRPbufferVI. ELECTROPHORESIS:Note:Addingsodiumacetatetothebottomreservoirduringelectrophoresis(SheenandSeed,1988,Biotechniques6:942-944)producesthesameeffectasrunningwedgeshapedgelsoraddingagradienttothegelsthemselves(Bigginet.al.,1983,PNAS80:3963-3965).Inallthreemethods,themobilityofsmall

28、DNAfragmentsisrestardedastheyapproachthebottomofthegel.Thesodiumacetatemethodissimplerthantheothermethods.1. Removeclamps.Cleanexcesspolyacrylamideandureafromthetopofplateassemblywithd.i.water.Pullthecomboutofthemoldslowlyandevenly,cleaningoutthecombareawithd.i.waterorbuffer.2. Addreservoirbuffersto

29、theapparatus.Thetopreservoirbufferis1XTBE.Thebottombufferreservoiris2/3XTBE,1Msodiumacetate.Make1500mlbottombufferforeachgel(100ml10 xTBE,900mld.i.water,500ml3MNaAcetate).3. Pre-electrophoresefor5-10mintowarmtheplatesothatthecombwilleasilyslideinplace.Cleanoutcombareawithbuffer.Topreventpossiblewell

30、towellleakage,applyaverylightcoatingofcellosealtotheoutsidesurfaceofthecomb,priortoinsertion.Placeplateassemblyingelboxandclamp.4. Formicrotiterformat,ahamilton8or12channelsyringeloadingdeviceforloadingthegelsisrecommended.Cleanouteachgroupofwellsimmediatelypriortoloadingwiththemultichannelhamiltons

31、yringe.Runthegelat50Cconstanttemperatureand100wattslimitingpowerforabout1.5-3hours,dependingonsizeofamplificationproduct.ConstanttemperaturecanbemaintainedwiththetemperatureprobeoptionoftheBioRadPower/Pak3000powersupply.VII. GELFIXING,STAININGANDCOLORDEVELOPMENT:Notesandtips:-Thisprocedureisadaptedf

32、romthePromegaSilverSequenceprotocol.-Usehighestqualityreagents.-Thefixer,stainanddeveloperaremadewith18mega-ohmwater(deionized=d.i.).-Thewashesaredonewithdistilledwater.-Weusepre-measuredsodiumthiosulfate(-20C)andsilvernitrate(RT),storedinmicrotubes.-Allincubationsandwashesareperformedatroomtemperat

33、ure,butthedevelopersolutionmustbepre-chilledto4-10Ctominimizebrownbackground.-Itisimportanttokeepthestopsolutionat4-10Caswell,forthesamereason.-Solutionscontainingformaldehydeshouldbehandledinafumehood.Theformaldehydeisaliquoted(RT)-Wearglovesthroughouttheprocedure.-Wearinganapronwillpreventsilverst

34、ainsonclothing.-Rockingplatformsarebetterthanorbitalshakerstoachieveevengelstaining.1. Freshlypreparestaininganddevelopersolutionswhilegelisrunning.Addformaldehyde,silvernitrateandsodiumthiosulfatetosolutionsimmediatelypriortouse.Topreparestainingsolution,combine2Ld.i.water,2gsilvernitrateand3ml37%f

35、ormaldehyde.Topreparedevelopingsolution,combine2Lchilledd.i.water,60gsodiumcarbonate,3ml37%formaldehydeand4mgsodiumthiosulfate.Chilldeveloperto4-10C.2. Removegelassemblefromrig,removesidespacersandcarefullyseparateglassplateswithaspatula.Thegelwillremainattachedtotheplatecoatedwithbindsilane.Cutofft

36、hetopcornerofthehighnumberedendofgelfororientation.3. Placegelandplateinashallowplastictraycontainingsufficientfreshfix/stopsolution(2liter7.5%glacialaceticacid)andgentlyagitatefor20minutesoruntiltrackingdyecompletelydisappears.WesometimesuseamultiplegelholderfromPromegaforprocessingseveralgelsatati

37、me.Twogelsrequirethesamevolumeofsolutionasone.Forthreegels,solutionvolumesareincreasedby50%.Note:Forovernightstorage,thegelcanbefixedasabove,rinsedwithd.i.waterandstoredinfreshfixerwithoutagitation.4.Rinsethegel3timesfor2minuteseachwithdistilledwaterwithgentleagitation.5. Addstainingsolutiontogeland

38、gentlyagitatefor30minutes,decantandprecipitatesilver.ThesilverintheusedstainingsolutionisprecipitatedwithNaCl(10g/2L)forrecycling.6. Rinsegelwithdistilledwaterfor5seconds(fromtimegelisplacedindistilledwatertotimeintodeveloper,i.e.aquickdip).Longerrinsesresultinweakersignal.Iftherinsegoestoolong,repe

39、atstep5withthestainingsolution.7. Transferthegelto800mlsofchilleddevelopingsolutionandgentlyagitatewitharockingmotionuntilthebandsfirstbecomevisible,usuallywithininafewminutes.Decanttheuseddeveloper,addtheremainingchilleddeveloperandcontinuetogentlyagitateuntilthebandshavereacheddesiredintensity.Over-developmentwillresultinabrownbackgroundwithlowcontrastbandsratherthanapencilgraybackgroundwithsharpbands.8.T