全 文 ：一种新的用于提高lea3基因扩增特异性和产量的增强剂*
于丽霞,武晓璐,汤晓倩,鄢暋波**
(西南林业大学园林学院,云南 昆明暋650224)
摘要:在对目的DNA序列尤其是高GC含量的片段进行PCR扩增的时候,经常需要对一些试验条件进行
优化。在一定条件下,二甲基亚砜、甲酰胺、甘油、NP灢40和Tween20等可以在某种程度上提高PCR的
特异性和效率。我们在对禾本科lea3基因进行分离克隆时发现了一种新的可以提高PCR产量和特异性的
物质———极高热稳定单链结合蛋白 (ETSSB),研究发现在每50毺lPCR反应体系中加入200ng的ET
SSB,可以有效地抑制DNA片段的非特异性条带的产生,并可以提高目的片段的产量。
关键词:高GC含量;聚合酶链式反应;极高热稳定单链结合蛋白
中图分类号:Q945,Q78暋暋暋暋暋文献标识码:A暋暋暋暋暋暋文章编号:0253灢2700(2010)06灢535灢04
EffectofNewSpecificityandYieldEnhancer
onAmplificationoflea3Gene
YULi灢Xia,WUXiao灢Lu,TANGXiao灢Qian,YANBo**
(FacultyofLandscapeArchitecture,SouthwestForestryUniversity,Kunming650224,China)
Abstract:AmplificationofatargetDNAbythepolymerasechainreaction(PCR)oftenrequiresexperimental
optimizationfactorsespecialywhenthetargetsequencecontainshighcontentofGC.Dimethylsulfoxide,
formamide,glycerol,NP灢40andTween20areknowntoincrease,thespecificityandefficiencyofPCRunder
certainconditions.Intheamplificationoflea3geneinGramineae,wefoundanewExtremeThermostable
Single灢StrandedDNABindingProtein(ETSSB)andimprovedyieldofPCR.Additionof200ngofETSSB
per50毺lreationdecreasedtheformationofnon灢specificDNAfragmentsandincreasedtheyieldofspecific
PCRproducts.
Keywords:HighGCcontent;PCR;ETSSB
暋 AnumberofPCRadditivesarenowcom灢
mercialyavailable,howevertheidentitiesof
theseagentsarenotusualyrevealedbytheir
suppliers.Frackmanetal灡(1998)havedemon灢
stratedthatBetaineisavailableatafractionof
thecostasa5mol·L灢1solution,andbesureto
useBetaineorBetaine(mono)hydrateandnot
BetaineHCl.DMSOat2%-10% maybenec灢
essaryforamplificationofsometemplates,how灢
ever10% DMSOcanreduceTaqpolymeraseac灢
tivitybyupto50% (Gelfand,1988).DMSOis
thoughttoreducesecondarystructureandis
particularlyusefulforGCrichtemplates.For灢
mamideisgeneralyusedat1%-5%and10%
formamideisreported(Gelfand,1989)tohave
noeffectontheactivityof Taqpolymerase,
however,Sarkaretal灡 (1990)foundthat1灡25%
formamideworkedaswelas2灡5%and5%,andno
云 南 植 物 研 究暋2010,32(6):535~538
ActaBotanicaYunnanica暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI:10灡3724/SP灡J灡1143灡2010灡10069
*
**
Foundationitems:TheAppliedFoundationResearchofYunnanProvinceinChina(Projectnumber2009ZC079M)andYunnan
ProvinceandMinisterKeySubject,UniversityKeyLabandSharePlatform
Authorforcorrespondence;E灢mail:yanbodr@yahoo灡com灡cn
Receiveddate:2010灢04灢01,Accepteddate:2010灢07灢02
作者简介:于丽霞 (1981-)女,助教,主要从事植物分子生物学研究。E灢mail:yulixia2005@gmail灡com
amplificationwasseenat10%soitseemsprudent
nottouseconcentrationsofformamidegreaterthan
strictlynecessaryforoptimalamplification.Non灢i灢
onicdetergentsstabilizeTaqpolymeraseandmay
alsosuppresstheformationofsecondarystruc灢
ture.0灡1%-1% TritonX灢100,Tween20or
NP灢40mayincreaseyieldbutmayalsoincrease
non灢specific amplification (Gelfand,1989).
TMAisgeneralyusedatafinalconcentrationof2
mmol·L灢1toeliminatenon灢specificpriming(Ko灢
varovaandDraber,2000).BSAhasprovenpar灢
ticularly useful when attempting to amplify
ancientDNAortemplateswhichcontainPCR
inhibitorssuchasmelanin.Andrecently,itwas
foundthatthePCRcouldbedramaticalyen灢
hancedbyAunanoparticles(Lietal灡,2005).
Single灢StrandedDNA BindingProtein (SSB)
keepsthestrandsseparatefromtendingtorevert
totheduplexformandalowingtheDNArepli灢
cationmachinerytoperformitsfunction.Single灢
strandedDNA (ssDNA)bindingproteinshave
beenassignedtheroleofremovingsecondary
structureinDNAandprotectingssDNAfrom
hydrolysisbynucleases (Chaseand Wiliams,
1986).However,inadditiontothesemundane
roles,ssDNAbindingproteinsarenowrecog灢
nizedasakeycomponentofthereplisomewhere
theyphysicalyandfunctionalyinteract with
otherreplicationproteinsandwiththeprimer灢
template(Benkovicetal灡,2001;O曚Donnel,
2006;HamdanandRichardson,2009).ssDNA
binding灢proteinsarealsoengagedin DNAre灢
combinationandrepair (LohmanandFerrari,
1994).Atpresent,SSBwasalsousedforRNA
targetstested(Goldmeyeretal灡,2007).Inview
ofthesemultiplerolesithasn曚tbeenreportedthatit
canremarkablyimprovetheamplificationofhigh
GCcontentsequencesespecialylea3gene.Inthis
research,itwasfoundthatitwasparticularlyuseful
forGCrichtemplates when200ngofExtreme
ThermostableSingle灢StrandedDNABindingProtein
(ETSSB)per50毺lreactionwasaddedinpolymer灢
asebufer,whichindicateditcouldremarkably
increasetheyieldandspecificityofPCRreac灢
tion.Mostofimportant,itwasconvenientand
efficientthataddingETSSBinnucleicacidam灢
plificationcomparingtootheradditives.
Group3LEA (Lateembryogenesisabun灢
dant)proteinsinclude11aminoacids(TAQAA灢
KEKAGE),resultingin amphypathic灢helixed
structureduringdehydrationofhigherplants
(Dure,1993).Unlikemostofeukaryoticgenes
whichcontainGC灢richsequencesmainlyinthe
promoterregion,GCcontentoflea3 geneis
higherinalcodingregion.
MaterialsandMethods
Amplificationreactionswerecarriedoutun灢
derthefolowingconditions:10mmol·L灢1Tris灢
HClpH8灡8,50mmol·L灢1KCl,1灡5mmol·L灢1
MgCl2,0灡2毺mol·L灢1primers,0灡5毺gtemplate,
0灡2mmol·L灢1eachdNTP,2灡5UTaqpolymer灢
ase.Thetemplates werecDNA andgenomic
DNAofbarley.TotalgenomicDNAwasisola灢
tedusingDNAquickPlantSystem.Taqpoly灢
meraseanddNTPwereobtainedfromTransGen
Biotech.PrimersweresynthesizedbyShanghai
Sangon BiologicalEngineering Technology &
ServiceCompanyLtd.Thesequencesofprimers
wereasfolows:forward,5曚灢atggcctccaaccagaac灢
cagg灢3曚,reverse,5曚灢ctagtgattcctggtggtggtgg灢3曚.
Additivereagentswereobtainedfromthefol灢
lowingsources.DMSO,formamide,glycerol
andTween20 werepurchasedfrom Amresco,
NP灢40werepurchasedfromFluko.ETSSBwas
purchasedfromBiolabs.
ThecyclingconditionsinaBiometer擬 TGra灢
dientthermalcyclewereaninitialincubationfor
5minat94曟folowedby35cycleseachof30s
ofdenaturationat94曟 and30sofannealingat
62曟for50sofextensionat72曟,afinalelonga灢
tionstepat72曟for7min.Amplifiedmaterials
weresubjectedtoelectrophoresisona1%agar灢
osegelstainedwithethidiumbromide,visual灢
izedonaUVPImagingSystemanddocumented
byVisionWorksLS.
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Recoveryofamplifiedfragmentswascon灢
ductedundermanufactorydescriptionofGelEx灢
tractionMiniKit(Huashun,Shanghai,China).
TheconnectionofTcarrierfromrecoveryadopts
pMD18灢TVectorSystems(TaKaRa,Dalian,Chi灢
na).ByDNAtransformationtechniques,trans灢
formedsusceptibilitycelswereculturedon50
mg·L灢1ampicilinplatefor9-16hunder37曟.
AftersinglecloneamplificationinLB,harvested
productusedforsequenceanalysisbyShanghai
ShenggongBiotechniqueCompany,Lt.
ResultsandDiscussion
Primersweredesignedaccordingtothere灢
portedsequencesofbarleylea3gene(GenBankID:
FJ026804).Ourpreliminaryexperimentsshowed
thatitwasverydificulttoamplifylea3genewith
genomicDNAofbarleyastemplatewithoutany
additive.Inotherpreviousresearch,DMSOor
combinationofbetaineandDMSOwasusedfor
theamplificationofsequenceswithhighGCcon灢
tent(Kangetal灡,2005).
Unlike most house灢keeping genes which
containCpGislandsintheirpromoterregion
thatarelimitedregionsofhigh灢densityCpGres灢
idues,theGCcontentofHVA1genewashigh.
However,wecouldamplifythetargetsequences
withoutany additive when positive Escherichia
coli.(DH5毩)strainincluding HVA1genewas
usedastemplate.Thus,weconsideredthatthead灢
ditivecouldimprovetheprobleminourresearchbe灢
cause:1.reducesecondarystructureandisparticu灢
larlyusefulforGCrichtemplates;2.additivespos灢
siblydecreasetheTm ofGCrichsequences;3.
protecttemplateDNAfromapurinic/apyrumidi灢
niceffectsofhightemperature;4.protectTaq
DNApolymerasefromhightemperature.
Inthispaper,itwasfoundthatET SSB
couldremarkablyimproveboththeyieldsand
thespecificityofamplificationofsequenceswith
highGCcontent,whileDMSOofconcentration
from3%to15%couldjustimprovetheyields.
AndtheyieldofPCRincreasedwhenthecon灢
centrationofDMSOchangedfrom3%to10%,
which wassimilarto Bookstein曚s,whilethe
productcouldbetestedevenwhentheconcen灢
trationofDMSOincreasedupto15%.Onlymi灢
nortargetproductscouldbeattainedinthepres灢
enceoflowconcentration(from1%to3%)of
formamide.Unlikethereportedthatglycerol
couldimprovetheamplificationofhigh(G+C)
templates,glycerolwiththeconcentrationfrom
1%to5% wasnothelpfultoamplifythefrag灢
mentsoflea3gene.Althoughnon灢ionicdeter灢
gentssuchas0灡1%-1% Tween20orNP灢40
arecommonlyusedinsampleprocessingproto灢
cols (Gelfand,1988),thesereagentshadno
contributiontoamplificationoflea3geneinour
experiment.AmongthecombinationsofDMSO
andglycerolatdifferentconcentration,itwas
foundthatonlycombinationof1%glyceroland
5% DMSO couldincreasetheyieldofproduct
slightly,otherscouldnotimprovetheamplifica灢
tionoflea3gene(Fig灡1).
Fig灡1暋TheeffectsofdifferentadditivesonPCR
M.DL2000marker;Lane1灢21Noneadditiveascontrol;2% DMSO;5% DMSO;10% DMSO;15% DMSO;20% DM灢
SO;1%formamide;2%formamide;3%formamide;1% glycerol;3% glycerol;5% glycerol;0灡2% Tween20;0灡8%
Tween20;0灡2% NP灢40;0.8% NP灢40;combinationof1%glyceroland5% DMSO;combinationof2%glyceroland5%
DMSO;combinationof1%glyceroland10% DMSO;combinationof2%glyceroland10% DMSO;4ng·毺l灢1ETSSB
7356期暋 暋暋YULi灢Xiaetal灡:EffectofNewSpecificityandYieldEnhanceronAmplificationoflea3Gene暋暋 暋暋
暋 Moreover,wehavesucceededinattaining
cDNAfragmentsoflea3genewithhighGCcon灢
tentfrom 68% to73% inseveralGramineae
plants including Ampelocalamus calcareous,
Sorghumv毺lgare,Cynodondactylon,Saccha灢
rumofficinarum,ZizanialatifoliandOryzaoffi灢
cinalis(Fig灡2),withacertainconcentrationof
ETSSB,fromwhichweconsideredthatETSSB
mightbehelpfultoalsequenceswithhighGC
content.
Fig灡2暋cDNAfragmentsoflea3genesindifferentplants
M.DL2000 marker;Lane1blankwithnoadditive;Lane2灢7 Ampelocalamuscalcareous,Sorghum
v毺lgare,Cynodondactylon,Saccharumofficinarum,ZizanialatifoliandOryzaofficinaliswithnoaddi灢
tive;Lane1blankwithET灢SSB;Lane9灢14Ampelocalamuscalcareous,Sorghumv毺lgare,Cynodondac灢
tylon,Saccharumofficinarum,ZizanialatifoliandOryzaofficinaliswithET灢SSB
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