SSR技术原理方法及步骤.docx
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SSR技术原理方法及步骤
SSR技术原理-方法及步骤
SSR技术
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,其中最常见是双核苷酸重复,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并计算等位基因频率。
在真核生物中,存在许多2-5bp简单重复序列,称为“微卫星DNA”其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
2)通过核酸数据库查询,从已有序列中搜寻包括SSR的序列并设计引物。
SSR分析实验的主要技术环节:
提取DNA;PCR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%胶只能分辨4-6bp差异);变性聚丙烯酰胺序列胶电泳;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—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并在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标记根据生物广泛存在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进行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BritishColumbia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/L
5、dNTP:
2.5mmol/L。
四、操作步骤:
1.在25ul反应体系中,加入
模板DNA1ul(30-50ng) ISSR引物1ul(约5pmol)
10xPCRBuffer2.5ul MgCl22ul
dNTP2ul Taq酶1单位(U)
加ddH2O至25ul 混匀稍离心,加一滴(约20ul)矿物油。
2.在PCR仪中预变性94℃2分钟,然后循环:
94℃1分钟,45℃-68℃40秒,72℃1-2分钟,共40轮循环。
3.循环结束后,72℃10分钟,4℃保存。
4.取PCR产物15ul加3ul上样缓冲液(6x)于1.6%或1.8%琼脂糖胶上电泳,稳压50-100V(电压低带型整齐,分辨率高)。
5.电泳结束,溴化乙锭染色20分钟。
6.用凝胶成像仪观察、拍照。
操作流程简图:
3.SSRGELandSilverStainingProtocol
PaulaMarquardt&CraigEcht
Publishedin:
EchtCS,May-MarquardtP,HseihM,ZahorchakR.1996.Characterizationofmicrosatellitemarkersineasternwhitepine.Genome39:
1102-1108.
CommentscanbedirectedtoPaulaMarquardtat:
USDAForestServiceResearch
5985HighwayK
Rhinelander,WI54501
USA
Phone:
715-362-1121
Fax:
715-362-1166
e-mail:
pmarquar@
I.EQUIPMENT:
DNAsequencingunit(35x45cm)&2000Vpowersupply
Clamps
Lg.plastictrays(4),about43x50x8cm,andonelid
Tworockingplatforms
Heatblockformicrotiterplates.Amicroplatevortexerishelpful.
II.MOLDASSEMBLY:
Notes:
Bindsilaneistoxicandshouldbeusedinachemicalfumehood.Weargloveswhenhandlingthissolution.Useasmallpieceofvinyltapeonaloweroutsidecorneroftheacryleasetreatedglassgelplatetomarktheuntreatedsideandalsohelpdistinguishtheplates.Thishelpsavoidconfusionbetweenplateswhenusingoffsetplates.Thetapecanremaininplacethroughseveralelectrophoresis/washingcycles.
1.Washinnerandouterplateswellwithalconoxcleanser.Rinsewellwithtapwater,deionizedordistilledwater,andethanol,airdry.Usededicatedspongesforeachtreatment.
2.Usingakimwipetissue,coattheinnersideofnotchedoroffsetplatewithacrylease(Stratagene)andallowtodry--Idonottreatthetop2inchesoftheplatesinceIfeelthatthenonstickcoatingpromotesleakingbetweenwellsbehindtheteethofthecomb.Buffwellwithakimwipesoakedinethanolforacleanfinish;thistakessomeelbowgreasetogetthestreaksoffoftheplate.Changeglovesbeforeworkingwithbindsilaneandtakecarenottocrosscontaminatetheplateswiththetwotreatments.Theacryleasetreatmentonlyneedstoberepeatedeveryfourgelsorso.
3.Preparefresh1mlbindingsolutionbymakingasolution0.0005-0.001%bindsilane(Sigma#M-6514)in95%ethanol,0.5%glacialaceticacid.Applywithakimwipeandcoattheinnersideofthelargerplatewithonemlandallowtodry4-5minutes.Wipetheplatewithethanolinonedirectionandthenperpendiculartofirstdirection,don"tusetoomuchpressure.Thistreatmentneedstoberepeatedeverytime.
III.GELSOLUTIONPREPARATION:
Note:
Acrylamideistoxic.Weargloveswhenhandlingsolutionandfacemaskwhenweighingoutpowder.Asaferalternativeistobuyapremix.
1.Rinseallglasswareandplasticwarewithd.i.waterpriortogelsolutionpreparationandpouring,includingthedisposablefilterunit.
2.Gelsare6%acrylamide,8Murea,1XTBE.Foreachgel,mixtogether50gurea,15ml40%19:
1acrylamidesolution,and31mld.i.water.Weusea4.5%gelforfingerprintingreactions.
Note:
(Westorealiquotsofpremixed40%Acrylogelsolution,Gallard-SchleisingerInd.,at-20℃.)
3.Warmandstirthemixtureinabeakerofwarmwateruntilalltheureaisdissolved.Add1.25gofamberliteresinandstir5min.Filterthrougha0.2uMfilteranddegasat25mgHgfor5min.Transfertograduatecylinderandadd10ml10xTBE,bringingvolumeto100mlwithd.i.water.
4.WehaverecentlystartedusingBurst-PackfromOwlScientific,whichisanacrylamidepremixincludingthebufferandcatalysts,forourfluorescentgelworkandareverypleasedwiththequalityandreproducibility.Thiswouldbeanoptionforthesilverstainingworkaswell.Theburst-pack"seliminatethechemicalweighingandmixing,deionizing,filteringanddegassingsteps.
IV.GELPOURING:
1.Immediatelypriortopouring,add500ul10%ammoniumpersulfate(0.1g+1mld.i.water)totheacrylamidemixinabeaker,gentlymixingwell.Thenadd50ulTEMEDandmix.PolymerizationwillnotstartuntilTEMEDhasbeenadded.Donotmixthecatalyststogetherbeforeaddingtothepolyacrylamidesolution--thiswillinhibitpolymerization.
2.WeusetheOtteradjustablegelcaster(OWLScientific,Inc.)forpouringgels.Inthissystemthegelispouredhorizontallywiththetopplateslidingoverthebottomplate,withouttheuseoftape,greaseorabottomspacer.
a.Placethelargerplateontocastersothatitabutstheendwallofthecaster.Moistenspacerswithwaterandplacethemflushtotheedgeofglassagainstthecasterwall.
b.Placethetopplate(notchedoroffset)sothatitstopedgeoverlapsthebottomedgeofthelowerplateby3-4cm.Usinga60mlsyringe,slowlydispensethegelsolutionbetweentheplates,allowingthesolutiontoflowbycapillaryaction.Gentlyslidethetopplateacrossthebottomplatewhiledispensingthegelsolutionalongtheleadingedgeofthetopplate.Ifanybubblesformwhilepouring,tryslidingthetopplatebacktouncoverthebubble,thenproceed.Amoreeffectivemethodistodragoutthebubbleswithaplastichook(freebyrequestfromPromegaCorp.)
3.Oncethegelispoured,inserttheflatedgeofasharktoothcomb(oracastingcomb)intothetopofthegeltothedepthdesiredforthewells.Place2-3clampsalongthesidesandtoptokeepplatesintightcontactwiththespacersandcombwhilethegelispolymerizing.
4.Allowthegeltopolymerizeatroomtemperature(RT)for1hour.GelcanbestoredatRTovernightifstepsaretakentopreventitfromdryingout.Todothis,placepapertowelsdampenedwithrunningbufferoverthetop(removeclampsbutleavecombinplace)andbottomedgesofthegelmoldandwrapwithplasticwrap.Donotstorethegelunderbuffer.
V.SAMPLEPREPARATION:
Notes:
Heatsamplesimmediatelypriortoloading.Keeptheloadingdyefresh.UseSSRPloadingdyethatislessthan2weeksold.Thedeionizedformamideusedinmakingtheloadingdyeshouldbelessthanonemonthold.
1.DenaturethesampleDNAbyadding1volume(10ul)offreshSSRPloadingdye(10mMNaOH,95%formamide,0.05%bromophenolblue,0.05%xylenecyanol)to1volumeofPCRsampleinamicrotiterplate.Mixwellandheatto95oCfor2min.Placeonice.2.MolecularweightstandardsarePGEM(Promega)andPoly-dA(Pharmacia#27-7836-01)sonicatedtoproducea1bpladder.PGEMisloadedinawellseparatefrompolyA.WedonotusepolyAforfingerprintinggels.Usinga144-well,microtiter4Xoffsetcomb,load3ulofthemix:
3.4ul1XPerkinElmerIIPCRbuffer
8.6ulof30ng/ulPGEM
12ulofSSRPbuffer
heat95℃for2min.,ice
and
5ul1XPEIIbuffer
2.5ulof400ng/ulofsonicatedPoly-dA
7.5ulSSRPbuffer
VI.ELECTROPHORESIS:
Note:
Addingsodiumacetatetothebottomreservoirduringelectrophoresis(SheenandSeed,1988,Biotechniques6:
942-944)producesthesameeffectasrunningwedgeshapedgelsoraddingagradienttothegelsthemselves(Bigginet.al.,1983,PNAS80:
3963-3965).Inallthreemethods,themobilityofsmallDNAfragmentsisrestardedastheyapproachthebottomofthegel.Thesodiumacetatemethodissimplerthantheothermethods.
1.Removeclamps.Cleanexcesspolyacrylamideandureafromthetopofplateassemblywithd.i.water.Pullthecomboutofthemoldslowlyandevenly,cleaningoutthecombareawithd.i.waterorbuffer.
2.Addreservoirbufferstotheapparatus.Thetopreservoirbufferis1XTBE.Thebottombufferreservoiris2/3XTBE,1Msodiumacetate.Make1500mlbottombufferforeachgel(100ml10xTBE,900mld.i.water,500ml3MNaAcetate).
3.Pre-electrophoresefor5-10mintowarmtheplatesothatthecombwilleasilyslideinplace.Cleanoutcombareawithbuffer.Topreventpossiblewelltowellleakage,applyaverylightcoatingofcellosealtotheoutsidesurfaceofthecomb,priortoinsertion.Placeplateassemblyingelboxandclamp.
4.Formicrotiterformat,ahamilton8or12channelsyringeloadingdeviceforloadingthegelsisrecommended.Cleanouteachgroupofwellsimmediatelypriortoloadingwiththemultichannelhamiltonsyringe.Runthegelat50℃constanttemperatureand100wattslimitingpowerforabout1.5-3hours,dependingonsizeofamplificationproduct.ConstanttemperaturecanbemaintainedwiththetemperatureprobeoptionoftheBioRad