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1、TheBACLIBRARYPROTOCOLSⅠ、MegaBaseDNAIsolationMegabase-sizeDNAisolationfromplantsToconstructlargeinsertDNAlibrariesinBACandYACvectors,methodsmustbedevelopedtoisolateveryhighmolecularweightDNA-megabase-sizeDNA–fromplants.ToisolatesuchDNA,protoplastsornucleimustfirstbeembeddedinagarose
2、plugsormicrobeads.TheagaroseactsasasolidyetporousmatrixwhichallowsforthediffusionofvariousreagentsforDNApurificationandsubsequentmanipulationswhilepreventingtheDNAfrombeingsheared(SchwartzandCantor,1984).Microbeadsarepreferredoverplugsbecausetheuseofbeadsincreasesthesurfaceareasurr
3、oundingthetissuesamplebyapproximately1000foldtherebyallowingformoreefficientandrapiddiffusionofchemicalsandenzymesintoandoutoftheagarosebeads(Cook,1984,OverhauserandRadic,1987,Wingetal,1993).Onceembedded,theprotoplastsornucleiarelysedandproteinsdegradedinthepresenceof0.5MEDTA,1%sar
4、cosyl,and0.1-1.0mg/mlofproteinase-Kat50℃(SchwartzandCantor,1984).Aftercelllysisandproteindegradation,theremainingDNAissuitableforenzymaticmodifications.Mostprotocolsfortheisolationofmegabase-sizeDNAfromplantsutilizetheprotoplastmethod(CheungandGale,1990,Ganal,etal,1989,Honeycutt,et
5、al,1992,Sobral,etal,1990,vanDaelen,etal,1989,Wing,etal,1993,Wooetal,1995).Althoughtheprotoplastmethodyieldsmegabase-sizeDNAofhighquality,theprocessiscostlyandlaborintensive.Forexample,toprepareprotoplastsfromtomato,youngleavesaremanuallyfeatheredwitharazorbladebeforebeingincubatedf
6、or4-5hourswithcellwalldegradingenzymes(GanalandTanksley,1989).Withsorghum,Wooetal(1995)foundthebestwaytogeneratehighyieldsofprotoplastsformegabase-sizeDNAisolationistorubcarborundumonbothsidesoftheleaveswithapaintbrush,50strokes/side,beforea4-5hourincubationwithcellulysin.Thustheam
7、ountoftimebeforeembeddinginagarosecanbebetween7-9hours,dependingontheamountofleafmaterialbeingpr4℃essed.Furthermore,sinceeachplantspeciesrequiresadifferentsetofconditionstogenerateprotoplaststhemethodwillonlyworkifahighyieldingprotoplastmethodhasbeendevelopedforagivenplantspecies.S
8、omeinvestigatorshavetriedw
