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Aone-tubemethodforrapidandreliableplantgenomicDNAisolationforPCRanalysisWeiHu,J.ClarkLagarias*DepartmentofMolecularandCellularBiology,UniversityofCalifornia,Davis,CA95616USA*Correspondingauthor,Emailjclagarias@ucdavis.edu

AbstractBackground:Consistent isolationofhighqualityplantgenomicDNAisaprerequisite for successful PCR analysis. Time consumption, ease ofoperation and procedure cost are important secondary considerationsfor selecting an effective DNA extraction method. The simple, reliableandrapidDNAextractionmethoddevelopedbyEdwardsandcolleaguesin1991[1]hasproventobethegoldstandard.Results:ThroughmodificationoftheEdwardsmethodofextraction,wehavedevelopedaone-tubeprotocolthatgreatlyimprovestheefficiencyof plant DNA extraction and reduces the potential for samplecontamination while simultaneously yielding high quality DNA suitableforPCRanalysis.WefurthershowthatDNAextractspreparedwiththismethodarestableatroomtemperatureforatleastthreemonths.Conclusion: The one-tube extraction method yields high quality plantDNAwith improvedefficiencywhilegreatlyminimizingthepotential forcross contamination. This low-cost and environment-friendlymethod iswidelyapplicableforplantmolecularbiologyresearch.

BackgroundPreparation of plant genomic DNA for PCR analysis is routinelyperformedinplantbiologyresearchlaboratories.AmongthemanyDNAextractionprotocolsintheliterature,themethoddevelopedbyEdwardsetal[1]isthemostwidelyused,asevidencedbyitsnearthreethousandcitations. The popularity of this method relies on its simplicity, thegeneration of consistently high quality DNA, and its reproducible PCRamplification.Althoughalternativeandmorerapidprotocolshavebeendeveloped, the quality of DNA is often sacrificed for the reduction inpreparationtimeusingtheseprotocols[2,3].FailedPCRanalysesarenotonlydisappointing,butoftenrequiretheresearchertorepeattheentireexperiment.Thus,thereliabilityoftheEdwardsmethodhasconsistentlyoutweighed the apparent time-saving benefits of other more rapidprocedures.ResultsandDiscussionTo improve the efficiency of the already simple and rapid Edwardsmethod for genomic DNA extraction for PCR analysis [1], we addedisopropanol directly with the crude tissue homogenate to precipitateDNAintheoriginalextractionvessel(seemethods).SincegenomicDNAcanberecoveredfrompelletedcellulardebris,thismodificationobviatesthe need for a new tube for isopropanol precipitation of DNA. In thisregard,thesupernatanttransferandlabelingofasecondarytubeintheEdwards method is a time-consuming step that has the potential formislabeling DNA samples when many samples are being preparedsimultaneously. This modification is thus environmentally friendly andavoids potential cross contamination, since a single tube is usedthroughout the extraction procedure. After the initial centrifugation, arapid air-dry method was found to be preferable to vacuum drying toremove traces of extraction buffer and isopropanol. The precipitatedgenomicDNAwasthenresolubilizedbyaddingwatertothegroundplantdebris.Theimprovedone-tubeextractionprotocoltakestenminutestoprocess two samples and is a considerable simplification of theexperimentalmanipulations.AsshowninFigure1,DNAextractedfromleavesofArabidopsisthalianaL.usingtheone-tubeprotocolwasamplifiedwithPCRefficiencysimilarto that extracted using the Edwardsmethod for < 2 kb amplicons.Our

extraction method was slightly less efficient for > 2 kb amplicons inroutinePCR.Theseresults indicatethatthedebrisdoesnotappreciablyinhibit PCR amplification of the extracted DNA.We also compared ourmethodwithaone-stepprotocoldevelopedbyKasajimaetal[3],whichisthemostrapidDNAextractionmethodavailable.DNAextractedbythismethodwaslessefficientlyamplifiedbyPCRforampliconssmallerthan2kb,andprovedunabletosupportamplificationof largeramplicons.Ourone-tubemethodprovedsuperiortothatofKasajimaetal.

Figure 1. PCR analysis of DNA extracts prepared using the standard Edwardsprotocol(1),theone-tubeprotocol(2and3)andtheone-stepprotocolofKasajimaetal(4).TwoindependentDNAextractsfrom(2)to(4)areshown;DNAextractsof(3)werestoredatroomtemperatureforthreemonths.To test the stability of DNA isolated by the one-tube method, DNAsolutionswerekeptonthebenchatroomtemperatureandanalyzedbyPCRover time.Figure1showsthatDNAextractscouldbestoredforatleastthreemonthsatroomtemperaturewithoutameasurablereductionin PCR efficiency compared to freshly prepared DNA extracts.Refrigeration or freezing is thus unnecessary for storage of DNAextractedby thismethod. This is particularly convenientwhenmultipleandsequentialPCRanalysesofthesameDNAsamplebecomenecessary.Forgroundplanttissuethatcontains largeamountsof fibrousmaterial,e.g.mature siliques, precipitated debrismay float up during prolongedstorage.Inthiscase,abriefcentrifugationwillre-establishthedebrisandDNAsupernatantphaseswithnolossofPCRamplificationefficiency.

Figure 2.DNAextractedbytheone-tubeprotocol iseffectivelyamplifiedby long-range PCR using LongAmp Taq DNA polymerase. Two independent DNA extractsusingEdwards(ED)andone-tube(OT)protocolsareshown.We further validated that DNA extracted by the one-tube methodpermits amplification of large DNA targets using long-range PCRtechnologywithanefficiencysimilartoDNApreparedwiththeEdwardsmethod(Figure2).Inparticular,atargeted~10kbfragmentwassteadilyamplified, indicating thatDNAof highmolecularweightwas preservedthroughout the extraction procedure. Figure 3 shows that reliable PCRamplifications of selected targets were achieved with DNA extractedfrom different Arabidopsis tissues using the one-tube method. Inaddition,thismethodworkedwellforawiderangeofleafbiomassinputfrom1to100mg(representingasinglecotyledonortworosetteleaves)

.CC-BY-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted February 14, 2020. . https://doi.org/10.1101/2020.02.13.948455doi: bioRxiv preprint

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with similar efficiency of PCR amplification. This indicates that theextractionvolumeof200µlused in theprotocol issufficientregardlessof the amount of tissue up to 100 mg. This is particularly helpful forextractionofpooledtissuesamplesfrommultipleplants.Grindingmorethan50mgofleafin200µlofEdwardsbufferisnoteasy,butstillyieldsreliable results,while5~10mgof leaf tissues is theoptimal range forbiomassinput.

Figure3.DNAextractsusingtheone-tubeprotocolfromvariousArabidopsistissuesandleafbiomasshavenegligibleinhibitoryeffectonPCRanalysis.Forleafbiomassandsizecomparison,1-2mgisapproximatelyequivalenttoonecotyledon;5mgisequivalenttotheleafmassexcisedusingtheinnercapofa1.5mlmicrocentrifugetube;25-50mgisequivalenttoonesinglefullydevelopedrosetteleaf.Wehaveemployedthisone-tubemethodforroutinePCRgenotypingofA. thaliana plants. Thismethodwas also successfully used for isolationandPCR analysis of genomicDNA from transgenic tobacco and tomatoplants(datanotshown).ConclusionsThe one-tube DNA extraction method described here significantlyreduces extraction time, while maintaining the same DNA quality (asjudgedbyPCRanalysis)comparedtothatoftheoriginalEdwardsmethod[1]. Moreover, despite the presence of cellular debris, one-tube DNAextractsarestableat roomtemperature forat least threemonths.Thismethodissimple,rapid,robust,inexpensiveandenvironmentallyfriendly.PlantbiologistcanbenefitfromthereductioninbothtimeandcostwhenusingthisDNAextractionmethodforPCRanalysis.MaterialsandMethodsDNAextractionThe one-tube plant DNA extraction protocol developed in this study isdescribedbelow.Fortheone-stepextractionprotocolofKasajimaetal.[3], the ground tissue solution was further centrifuged briefly toprecipitatedebris,whichwe found to improve thesuccess rate forPCRreactions.One-tubeDNAextractionprotocol1. Collect~5-15mgofleaftissueintoa1.5mlmicrocentrifugetube;2. Add 200µl Edwards buffer [1], grindwellwith a plastic pestle (< 20

sec);3. Add200µlisopropanolandmixwellbygentleinversion;4. Centrifuge13,000rpmx5min;5. Decantthesupernatant,invertthetubeonpapertowertoairdrythe

pellet(<2min);6. Add100µlofsteriledeionizedH2O,dragthetubethroughatuberack

toquicklysuspendthepellet(moreeffectiveandeasierthanvortex);7. Centrifuge13000rpmx1mintoprecipitateinsolubledebris;8. Take1-1.5µlofsupernatantfor20µlofPCRanalysis.

DNA solutions extractedwith the abovemethod are stable for at leastthree months at room temperature and at least one year at 4⁰C forreliablePCRanalysis.PrimersusedinthisstudyPrimersforPCRanalysesprimingtheA.thalianaPHYBgenewereoWH13(5'-CAACAGCGGGAACAATGAAATG-3'), oWH14 (5'-TGAACGCAAATAATCTCCCTCTTC-3'),oWH31(5'-TGTTTAAGCAGAACCGTGTCCG-3'),oWH32(5'-TTCCATCCATTGATGCAGCCTC-3'), oWH105 (5'-GCGATTGGTGGCCAAGAT-3'), oWH130 (5'-TGATTCACCCTAAATCCTTCCTTG-3'), and oWH131 (5'-TGCTGTGTGCGGTATGGCAG-3'). oWH131 and oWH32 were foramplifyinga438bpfragment,oWH31andoWH32for1099bpfragment,oWH105 and oWH14 for 1542 bp fragment, oWH130 and oWH32 for2109 bp fragment, and oWH130 and oWH13 for 3535 bp fragment,respectively. The primers used for long-range PCR were chosen forpriming a genomic PHYBY276H transgene allele (YHBg/phyA-201 phyB-5Line #5, [4, 5]); they were oWH130 and oWH197 (5'-TGCTCTAGCATTCGCCATTCAG-3') for amplifying 4482 bp fragment,oWH130andoWH40 (5'-CACCGACTACGCTTCACAGAAAG-3') for7300bpfragment,andoWH120(5’-TGAAGGCGGGAAACGACAATC-3')andoWH40for9874bpfragment.PCRamplificationandgelelectrophoresisFor routine PCR, 1 ~ 1.5µl of DNA extractwas added to 20µl of PCRreaction mix containing standard PCR buffer and 1 unit of Taq DNApolymerase (Cat # = M0273, New England Biolabs, Beverly, MA). Forlong-rangePCR, 1.5µl ofDNAextractwas added to25µl of LongAmpTaq DNA polymerase PCR reaction mix (Cat # = M0323, New EnglandBiolabs) following the manufacturer’s protocol. PCR reactions wereperformed using a DNA Engine thermal cycler (Bio-Rad, Hercules, CA)with optimized annealing temperature for 35 ~ 40 cycles. All 20 µl ofroutinePCRproductsand2.5µloflong-rangePCRproductswereloadedfor 1%agarose gel electrophoresis.Gel imageswere inverted inAdobePhotoshop.Authors’contributionsW.H. designed and performed all experiments; J.C.L. provided funding,facilities and supervision. Both authors have read, corrected andapprovedthefinalversionofthemanuscript.AcknowledgementsWethankTimothyButterfieldforcriticalreadingofthemanuscript.ThisworkwassupportedbygrantGM068552fromtheNationalInstitutesofHealth.References1. EdwardsK,JohnstoneC,ThompsonC:Asimpleandrapidmethodfor

thepreparationofplantgenomicDNAforPCRanalysis.NucleicAcidsRes1991,19(6):1349.

2.WangH,QiM,CutlerAJ:AsimplemethodofpreparingplantsamplesforPCR.NucleicAcidsRes1993,21(17):4153-4154.

3.KasajimaI,IdeY,Ohkama-OhtsuN,HayashiH,YoneyamaT,FujiwaraT:Aprotocol for rapidDNAextraction fromArabidopsis thaliana forPCRanalysis.PlantMolecularBiologyReporter2004,22:49-52.

4. Su YS, Lagarias JC: Light-independent phytochrome signalingmediatedbydominantGAFdomaintyrosinemutantsofArabidopsisphytochromesintransgenicplants.PlantCell2007,19(7):2124-2139.

5. HuW,SuYS,LagariasJC:Alight-independentalleleofphytochromeBfaithfully recapitulatesphotomorphogenic transcriptionalnetworks.MolPlant2009,2(1):166-182.

.CC-BY-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted February 14, 2020. . https://doi.org/10.1101/2020.02.13.948455doi: bioRxiv preprint