chapter  4
12 Pages

ISOLATION AND CHARACTERIZATION OF PLANT DNAs

TheabilitytoextractandanalyzeDNAfromplanttissuesisanessentialaspectofplant

molecularbiologyandmaybedividedintotwostages.Inthefirststage,plantcellsare

disruptedtoreleasetheDNA.Thisisacriticalstage.inthatfailuretodisruptallofthecells

inthetissueortoprotecttheDNAduringdisruptionmaygreatlyreducethefinalyieldof

DNA.ItisatthispointthatadecisionmustbemadewhethertoisolatetotalDNAortoobtain aspecificDNAfractionfromthenucleus,chloroplast,ormitochondria.TotalDNAis

acceptableforanalysisofnucleargeneswithoutcloselyrelatedorganellargenes.orfor

organellargeneswithoutcloselyrelatednuclearhomologues.Fractionationofthenuclearand

organellargenomesmaybeusefulforassigningagenetoaparticulargenome.foravoiding

cross-hybridizationofrelatedgenes,andforgeneratingorganelle-specificlibrariesforclon-

ing. Inthesecondstage,theDNAisextractedawayfromtheothercellularconstituentssuch

asproteins.carbohydrates,membranes.andcellwalls.Itisimportantatthisstagetoensure

thatsignificantamountsofDNAarenottrappedinthecelldebris,andthattheDNAis

completelydissociatedfromproteinsandothercontaminantsthatmightcopurifyandinterfere

withsubsequentanalyses. AnumberofdifferentmethodsforpurifyingplantDNAhavebeendescribed. 1·7Itis

beyondthescopeofthischaptertoconsidereachmethodandcomparerelativeadvantages

anddisadvantages;however.somegeneralconsiderationsthatmightbeappliedwhenevalu-

atingaparticularmethodare:

I.Willitworkwithmytissue?Sometissueshavehighlevelsofnucleaseorpolyphenol

oxidase.DNAisolationfromthesetissuesmayrequireadditionalstepsandprecautions notnecessarywithmoretractabletissues.