Molecular colog! "esources #200$% 9& '()'* doi+ (0,((((-.,(/**00$$8,2008,02(8',1 TECHNICAL AD VA NCES An inexpensive high-throughput method to extract high ie!ds o" good #ua!it DNA "rom "ungi T, 2 3 2 4 5 6 3 & 7 , 2 A " 3 M & 6 , M A 8 4 9A & 9, : TA and T, 2 4 B : 7 : Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 3058687, Japan A$stract %e deve!oped an e""icient method "or high-throughput extraction o" high-#ua!it DNA "rom various "ungi& In this method' "unga! mce!ia (ere cu!tured and harvested on the sur"aces o" mem$ranes on media p!ates& %e degraded ce!! (a!!s using a !tic en)me *+ata!ase,& -uri"ication (as per"ormed on 9.-(e!! g!ass "i$re "i!ter p!ates& DNA (as success"u!! extracted "rom various "ungi provided */01 genus /21 species, at high ie!ds and #ua!it' and proved suita$!e "or storage' po!merase chain reaction amp!i"ication and restriction en)me digestion& The method descri$ed is rapid' inexpensive and automation "riend!& This ena$!es the simu!taneous extraction o" !arge num$ers o" samp!es' signi"icant! improving the potentia! throughput in genomics' particu!ar! in diagnostic and popu!ation studies& ey!ords+ ;ilamentous ;ungi& genomic <7A& high0throughput method& P5"& 9atalase Recei"ed #$ %ctober #007& re"ision accepted '3 February #008 Introduction 1traction o; genomic <7A ;rom ;ilamentous ;ungi is labourious and time0consuming, =ilamentous ;ungi ha>e e1tremel! hard cell walls and possess pol!saccharide and pol!phenolic compounds& which are di;;icult to remo>e and can strongl! inhibit reactions integral to <7A anal!sis #Al08amarrai ? 8chmid 2000%, 5on>entional methods ;or <7A e1traction rel! on the mechanical disruption o; m!celia using grinders #with or without li@uid nitrogen% to reduce the plasticit! o; the cell wall& and re@uire cet!ltrimeth!l ammonium bromide #5TAB% and phenol)chloro;orm to remo>e pol!saccha0 ride and pol!phenolic compounds #>an Burik et a(, ($$8%, These methods are time0consuming and cannot be auto0 mated& thereb! precluding their usage in high0throughput pro.ects, 5hele10based protocols are a @uick and low0cost option& but <7A e1tracted in this wa! is generall! low !ield and unstable #Manian et a(, 200(%, Man! other rapid e1traction methods ;or pol!merase chain reaction #P5"% ampli;ication ha>e been reported #5enis ($$2A 6augland et a(, ($$$A Liu et a(, 2000A 2arakousis et a(, 200BA 8uCuki et a(, 200B%, Although these methods enable @uick P5"0based anal!ses& 5orrespondence+ Taisei 2ikuchi& =a1+ 8( 2$ 8/D(*'DA 0mail+ kikuchitEa ;;rc,go,.p 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd the! are unsuitable ;or obtaining large amounts o; pure <7A ;or anal!ses like 8outhern blots and ampli;ied ;ragment length pol!morphism #A=LP%& as well as ;or storage, 8e>eral commercial <7A e1traction kits can success;ull! be used with ;ungi #Fri;;in et a(, 2002%, Most o; these products use silica0based methods and deli>er ;aster& more stable and more sensiti>e <7A e1tracts, 6owe>er& the cost o; these kits remains high and is o;ten prohibiti>e ;or large0scale anal!sis, 6ere& we present a new method utiliCing enC!matic l!sis o; the cell wall and glass ;ibre0based puri;ication o; <7A using $B0well plates, This method is rapid& ine1pensi>e and automation ;riendl!& and can produce high !ields o; good @ualit! <7A in a high0throughput manner, 3ateria!s and methods Rea)ents and supp(ies The reagents are readil! a>ailable as standard components& such as Tris& <TA& 7a5l& ethanol and 20 propanol& or as premade solutions& such as bu;;er AP2 #G3AF7%, He chose the AcroPrep $B =ilter Plate #Pall% as our ;ibre ;ilter plate ;or binding genomic <7A, The n!lon membrane used was Biod!ne A 8* mm disc #Pall%, 5ellophane membrane was purchased at a stationer& cut into s@uare pieces o; * I * cm& washed in water and autocla>ed, 41 T5673 5AL A<JA 758 Ta$!e / =ungal species ;or <7A e1traction Ascom!cetes *isce((a castanea +oni(ia kusanoi Rhi,osphaera sp, -.phiporthe ra"ene(iana *othidea sp, +onochaetia .onochaeta /area resinae -p(ospore((a sp, *othiore((a sp, +onostiche((a sp, /c(erotinia sc(erotioru. -rthriniu. phaeosper.u. *repanope,i,a puncti0or.is +ycosphaere((a (u,onensis /e(enopho.a sp, -scochyta pisi 1ndothia radica(is +ycosphaere((a .yricae /eptotis sp, -sperisporiu. se2uoiae 1ndothie((a sp, +ycosphaere((a to)ashiana /phaeropsis crypto.eriae 3arta(inia robi((ardoides 1nto.osporiu. sp, +y4osporiu. rhois /phaeropsis sapinea 3otryosphaeria dothidea 1picoccu. ni)ru. +y4osporiu. sp, /ta)onospora cinna.o.u. 3otrytis cinerea 1picoccu. sp, 5ectria cinnabarina /ta)onospora sp, 6a(onectria kyotensis Fusariu. o4ysporu. 5ectria sp, /trasseriopsis tsu)ae 6enan)iu. 0erru)inosu. Fusariu. 0u7ikuroi 5eocos.ospora sp, Taphrina !iesneri 6enan)iu. sp, 8ibbere((a baccata %phiosto.a piceae Trichotheciu. roseu. 6eratocystis .inor 8(ioc(adiu. sp, %phiosto.a sp, Trichoder.a (on)ibrachiatu. 6ercospora popu(ico(a 8(oeosporiu. ka!aka.ii Pate((ina sp, Trochophora 0ascicu(ata 6ercospore((a 7u)(andis 8(oeosporiu. sp, Penici((iu. citrinu. Truncate((a sp, 6haeto.iu. 0unico(a 8no.onia setacea Penici((iu. indonesiae Tubercu(ispora sp, 6haeto.iu. )(obosu. 8ui)nardia a(ni)ena Penici((iu. .e)asporu. 9a(sa kun,ei 6haeto.iu. sp, 8ui)nardia (aricina Penici((iu. pseudostro.aticu. 9a(sa ni"ea 6(adosporiu. sp, :a(bania 7uniperi Penici((iu. "errucosu. 9a(saria sp, 6o((etotrichu. coccodes :endersonia sp, Periconia a,a(eae 9ertici((iu. sp, 6o((etotrichu. truncatu. ;achne((u(a resinaria Pesta(otia aceris <ythia sp, 6orynespora cassiico(a ;epteutypa cupressi Pesta(otia popu(i=ni)rae 6oryneu. sp, ;epto)raphiu. (undber)ii Pesta(otiopsis )(andico(a Basidiom!cetes 6ryphonectria parasitica ;epto)raphiu. proceru. Pesta(otiopsis )uepini -.y(ostereu. areo(atu. 6ryptodiaporthe castanea ;epto)raphiu. !in)0ie(dii Phaeoseptoria euca(ypti 14obasidiu. bisporu. 6y(indrosporiu. sp, ;eptosphaeria sp, Phia(ophora sp, F(a..u(ina "e(utipes *iaporthe .edusaea ;eucosto.a persoonii Pho.a sp, :e(icobasidiu. .o.pa *iaporthopsis sp, ;ophoder.iu. pinastri Pho.opsis rudis :e(otiu. (euce((u. *iatrype sp, +acropho.a 2uercico(a Phy((osticta a(cides +undkure((a ka(opanacis *iatrype((a sp, +acropho.a su)i P(ectosphaera crypto.eriae Thanatephorus cucu.eris *icarpe((a dryina +acropho.ina phaseo(ina Racodiu. therryanu. *idy.e((a sp, +e(anconis 7u)(andis Ra00ae(ea 2uerci"ora *ip(ocarpon .espi(i +e(anconis sti(bosto.a Retinocyc(us sp, *ip(odia sp, +e(anconiu. ob(on)u. Rhi,ina undu(ata *ip(odina popu(i +etasphaeria sp, Rhi,osphaera ka(kho00ii The 9atalase l!sis solution was composed o; (K 9atalase #Ta2a"a%& 200 Lg-mL "7ase A #3n>itrogen%& (0 mm sodium phosphate #p6 B%& 0,8 M 7a5l& and *0 mm <TA #p6 /%& and was steriliCed b! ;iltration be;ore use, The 8<8 l!sis solution was composed o; 8K 8<8& 0,* mg-mL proteinase 2& (00 mm 7a5l& (0 mm Tris #p6 8%& and ( mm <TA, /a.p(e preparation The ;ungal strains used in this stud! were ;rom the culture collection stored within the =orest Patholog! Laborator! at the =orestr! and =orest Products "esearch 3nstitute o; Japan, =ungi were cultured on cellophane membranes placed on potato de1trose agar #P<A& iken 5hemical% plates at 2D M5 ;or periods appropriate ;or each ;ungus, =ungal m!celium was har>ested ;rom each plate into (,*0mL tubes or the wells o; $B0deep0well plates b! scratching the sur;ace o; the membranes using steriliCed small metal spatulas, 3n each case& the m!celium was stored at )80 M5 T5673 5AL A<JA 758 42 ;or ;urther <7A e1traction, He used n!lon membranes in place o; cellophane membranes onl! when it was di;;icult to har>est m!celium because o; se>ere degradation o; cellophane membranes during ;ungal culti>ation, To test this method& we used m!celial samples #()D0 mg% o; a >ariet! o; ;ungi ;or <7A e1traction& including (2* species o; ascom!cetes and / species o; basidiom!cetes #Table (%, *5- puri0ication <7A puri;ication was per;ormed as ;ollows+ / 9atalase l!sis solution #200 LL% was added to each tube or well and incubated at D/ M5 ;or 0,*)( h with agitation at (000 r,p,m, using a shaking incubator #32(00J& 9amato 8cienti;ic 5o%, 1 8<8 l!sis solution #B/ LL% and (00 LL o; Circonia beads #0,* mm diameter% were added to each tube or well& >orte1ed ;or ( min and incubated at B0 M5 ;or (0 min, 2 8olution AP2 #8/ LL% was added to each tube or well, A;ter incubation on ice ;or * min& samples were centri;uged at (* 000 g ;or * min #(,*0mL tubes% or D000 g ;or (* min #$B0 deep0well plates% to pellet the debris, 4 Be;ore trans;erring ('0 LL o; the cleared l!sate to a $B0 well glass ;ibre ;ilter plate& 220 LL o; 20propanol was dispensed into each tube o; the plate, 5 A;ter a static *0min wait to ;acilitate binding o; the 20 propanol0precipitated <7A to the glass ;ibres& >acuum pressure was applied using a Millipore >acuum mani0 ;old to remo>e the cell l!sate solution, . The glass ;ibre ;ilters were then washed ;our times with 200 LL o; 80K a@ueous ethanol b! >acuum ;iltration, 6 =ilter plates were centri;uged at D000 g ;or D min to remo>e residual ethanol and dried using a >acuum e>aporator, 7 Plates were resuspended with B0 LL o; preheated #B* M5% T #(0 mm Tris)65l& p6 8,0& 0,( mm <TA%& allowed to stand ;or * min and then eluted b! centri;ugation at D000 g ;or * min into a $B0well collection plate, This elution step was repeated once& resulting in (20 LL o; ;inal elution >olume, P6R a.p(i0ication The large subunit region o; ribosomal <7A was ampli;ied using P5" primers nu0L84000'2)*N #L"0"% #A555F5TFA0 A5TTAAF5% and nu0L8400$'8)DN #L"*% #T55TFAFFF0 AAA5TT5F% #a location map and oligonucleotide se@uences o; these primers can be ;ound at ww w ,biolog ! ,duke,edu- ;ungi-m!colab-primers,htm%, P5" ampli;ications were carried out in D00LL reaction mi1tures containing (* LL FoTa2 Freen Master Mi1 #Promega%& 0,* Lm o; each primer and ( LL o; each <7A e1tract, 5!cling conditions were $' M5 ;or ( min& then D0 c!cles o; $' M5 ;or D0 s& *D M5 ;or D0 s and /2 M5 ;or ( min, *i)estion !ith restriction en,y.es "estriction enC!me digestion was per;ormed in D00LL reaction mi1tures containing the appropriate bu;;er #(I%& 20 4 o; each restriction enC!me #3a.63& 1co"3& :ind333 or Pst3% and (0 LL o; each <7A e1tract, To assess the auto0 degradation o; <7A& a <7A sample was also incubated under the same conditions but in absence o; restriction enC!me, 8esu!ts and discussion Hith this method& <7A was success;ull! e1tracted ;rom >arious ;ungi e1amined #(D2 species% #Table (%, <7A @ualit! was tested b! direct electrophoresis, 6igh molecular weight <7A was obser>ed when * LL o; the <7A solution was electrophoresed on (K agarose gel and >isualiCed b! ethidium bromide staining #=ig, (%, 9ig& / Agarose gel #(K% electrophoresis o; e1tracted <7As ;rom (B species o; ;ungi, =i>e microlitres o; e1tracted solutions was loaded, M& 20log ladder #0,* Lg%A (& 6eratocystis .inorA 2& ;epto)raphiu. !in)0ie(diiA D& +onochaetia .onochaetaA '& ;epteutypa cupressiA *& +ycosphaere((a to)ashianaA B& +ycosphaere((a (u,onensisA /& %phiosto.a piceaeA 8& %phiosto.a sp,A $& Pesta(otiopsis )uepiniA (0& Pesta(otiopsis )(andico(aA ((& Phaeoseptoria euca(yptiA (2& Rhi,ina undu(ataA (D& Retinocyc(us sp,A ('& /area resinaeA (*& Penici((iu. indonesiaeA (B& Penici((iu. pseudostro.aticu., 9ig& 1 P5" ampli;ication o; ribosomal <7A o; (B species o; ;ungi, The large subunit o; ribosomal <7A was ampli;ied and * LL o; P5" products was loaded on a (K agarose gel, 7umbers correspond to the species listed in =ig, (, <7A concentrations were (0)(00 ng-LL #appro1imatel! (,2)(2 Lg o; <7A in total elution% and 2B0-280 ratios were (,B)(,$ when measured b! 7ano<rop spectrophotometr! #7ano<rop Technologies%, 3n the case o; one representati>e species& Ra00ae(ea 2uerci"ora& we obtained appro1imatel! ( Lg o; <7A ;rom (0 mg o; m!celium sample with this method, This amount was comparati>el! high& indicating the pre0 sented method is e;;icient ;or obtaining pure <7A ;rom ;ungi, Moreo>er& <7A e1tracts were used as templates ;or P5" ampli;ication o; the large subunit region o; ribosomal <7A, An appro1imatel! $000bp ;ragment was success;ull! ampli;ied ;rom each <7A e1traction #=ig, 2%, =urthermore& <7A e1tractions were e;;icientl! digested with restriction enC!mes #=ig, D%& while no degradation was obser>ed in e1tracted <7A incubated under the same condition without restriction enC!mes #data not shown%& impl!ing that the @ualit! o; the <7A e1tracted b! this method was su;;icient ;or digestion b! restriction enC!mes, He suggest that <7A puri;ied b! the presented method is o; such high @ualit! 9ig& 2 <igesti>it! o; <7A prepared with the presented method, Ten microlitres o; e1tracted <7A was digested with 3a.63 at D/ M5 ;or D h in D00LL reaction mi1ture and B LL o; the mi1ture was loaded on a (K agarose gel, 7umbers correspond to the species listed in =ig, (, 8imilar electrophoresis patterns were obtained when <7A was digested with 1co"3& :ind333 or Pst3 >not shown%, and !ield that it can be used not onl! ;or P5" ampli;ication but also ;or anal!ses that re@uire large amounts o; high @ualit! <7A& such as 8outhern blot or A=LP anal!sis, 3n addition& <7A @ualit! a;ter storage at ' M5 ;or B months did not diminish& measured b! electrophoresis images and the le>el o; P5" ampli;ication #data not shown%, He belie>e @ualit! o; entire set o; <7A will remain intact a;ter storage ;or more longer period o; time, 3n this stud!& we demonstrated a new method ;or high0 !ield& high0@ualit! <7A e1traction ;rom >arious ;ungi, This e1traction and puri;ication procedure is likel! to be applicable ;or broad ranges o; conditions& including a wide breadth o; the 2ingdom =ungi, Hith this method& <7A was e1tracted ;rom >arious ;ungal tissues including tissues that ha>e been preser>ed in absolute ethanol& spores and ;resh& ;roCen or dried ;ruit bodies, <7A were readil! e1tracted ;rom all samples and electrophoresis images o; e1tracted <7A were similar to those o; <7A e1tracted using commercial kits #<7eas! Plant mini kit& G3AF7% #data not shown%, 3t is widel! accepted that some ;ungi ha>e highl! acti>e nucleases& which pre>ent e1traction o; high molecular weight <7A, 3n con>entional methods& harm;ul protein denaturants such as phenol or guanidine thioc!anate are used to inacti0 >ate the nucleases, Because we did not use these protein denaturants in this method& degradation o; <7A during the e1traction process ma! ha>e occurred, 6owe>er& we obtained high molecular weight <7A ;rom all ;ungi used, <7A degradation might be minimiCed b! handling the ;roCen samples @uickl! so that the! cannot be dissol>ed be;ore addition o; l!sis bu;;er containing *0 mm <TA, Flass substrates #particles& beads and ;ibres% ha>e been used ;or puri;!ing nucleic acids under >arious conditions& including <7A in agarose gels #Jogelstein ? Fillespie ($/$%& plasmid <7A in bacteria #<e derich et a(, 2002% and genomic <7A in animal or plant tissues #3>ano>a et a(, 200B%, Binding o; <7A to glass substrates is generall! ;acilitated b! the presence o; high concentrations o; chaotropic salt such as sodium iodide and sodium perchlorate #Jogelstein ? Fillespie ($/$A 6oarau et a(, 200/% or b! the presence o; high concentrations o; sodium chloride and pol!eth!lene gl!col #PF% #ngelstein et a(, ($$8%, These methods ha>e the common element o; high salt solutions& in which the adsorption o; plasmid <7A onto the glass substrate occurs most likel! b! a mechanism similar to adsorption chroma0 tograph! #<e derich et a(, 2002%, 6owe>er& unlike the high salt methods& we used 20 propanol to precipitate the <7A onto glass sur;ace, This method has been success;ull! used to puri;! plasmid <7A ;rom 1scherichia co(i #<e derich et a(, 2002%, 3n this stud!& we showed the method can be used ;or genomic <7A preparation ;rom ;ungi& as well, 3n ;act& our newl! de>eloped method o;;ers some de;inite ad>antages o>er traditional methods o; ;ungal <7A e1traction, =irst& m!celium used ;or <7A e1traction was collected ;rom the sur;aces o; cellophane or n!lon mem0 branes on P<A plates, 3n con>entional methods& li@uid culture was ;re@uentl! used to grow ;ungi be;ore <7A e1traction, 3n this method& b! culturing on membranes on P<A plates& we reduced the risk o; media contamination& which sometimes inhibits subse@uent enC!matic reactions, Additionall!& the risk o; bacterial or ;ungal contamination can also be minimiCed because the colonial morpholog! o; each ;ungus can be >isuall! checked on the plate be;ore collection, 8econd& a l!tic enC!me #9atalase% was used to destro! cell walls o; the ;ungi, This replaced the traditional mechanical disruption step& which is labourious due to the di;;icult! in disruption o; ;ungal cell walls b! brie; mechanical methods such as bead mills or sonication& which also incurs signi;icant losses when mortars and pestles are used ;or disruption, nC!matic l!sis is non0 laborious and enables the procedure to be completed in one tube, =inall!& the e1traction and puri;ication steps were accomplished with $B0well plates& which reduce the labourious handling o; multiple samples& are automation ;riendl! and allows ;or e;;icient downstream processing, Thus& our de>eloped method achie>es great sa>ings in both time and cost, The e1traction o; $B samples ;rom m!celia in tubes-wells can be completed within D h, The appro1imate cost o; each <7A e1tract prepared b! the present method is O P0,** making it si1;old cheaper than commerciall! a>ailable kits, The o>erall picture emerging ;rom this stud! is the de>elopment o; rapid& ine1pensi>e and reliable method ;or the e1traction o; genomic <7A ;rom ;ungi, To understand the ;ungal genetics and their ecological relationship& it is necessar! to obtain a high !ield o; ;ungal <7A with good @ualit!, The high0throughput e1traction method designed and de>eloped in this stud! will pro>ide a better opportunit! ;or complete and precise anal!sis o; ;ungal genomes in a wa! to understand the molecular ecolog! o; >arious kinds o; ;ungi, Ac:no(!edgements The authors thank Asuka 8hichiri ;or technical assistance, This work was ;unded b! the Japanese Ministr! o; ducation& 8cience& 8ports and 5ulture& Frant0in0Aid ;or ncouragement o; 9oung 8cientists #B% (8/800D2 #T2%& (///00/* #9:% and Frant0in0Aid ;or 1plorator! "esearch (8B*/0D2 #6M%, 72 is supported b! Japan 8ociet! ;or the Promotion o; 8cience #J8P8% Postdoctoral =ellowship ;or ;oreign researchers, 8e"erences Al08amarrai T6& 8chmid J #2000% A simple method ;or e1traction o; ;ungal genomic <7A, ;etters in -pp(ied +icrobio(o)y& 20& *D)*B, >an Burik JA& 8chreckhise "H& Hhite T5& Bowden "A& M!erson < #($$8% 5omparison o; si1 e1traction techni@ues ;or isolation o; <7A ;rom ;ilamentous ;ungi, +edica( +yco(o)y& 2.& 2$$)D0D, 5enis JL #($$2% "apid e1traction o; ;ungal <7A ;or P5" ampli;ication, 5uc(eic -cids Research& 10& 2D80, <e derich <A& :kwuonu F& Farner T et a(, #2002% Flass bead puri;i0 cation o; plasmid template <7A ;or high throughput se@uencing o; mammalian genomes, 5uc(eic -cids Research& 20& eD2, ngelstein M& Aldredge TJ& Madan < et a(, #($$8% An e;;icient& automatable template preparation ;or high throughput se@uencing, +icrobia( and 6o.parati"e 8eno.ics& 2& 2D/)2'(, Fri;;in <H& 2ellogg 5A& Peak 22& 8hinn A #2002% A rapid and e;;icient assa! ;or e1tracting <7A ;rom ;ungi, ;etters in -pp(ied +icrobio(o)y& 24& 2(0)2(', 6augland "A& 6eckman JL& H!mer LJ #($$$% >aluation o; di;;erent methods ;or the e1traction o; <7A ;rom ;ungal conidia b! @uantitati>e competiti>e P5" anal!sis, Journa( o0 +icrobio(o)ica( +ethods& 26& (B*)(/B, 6oarau F& 5o!er JA& 8tam HT& :lsen JL #200/% A ;ast and ine1pen0 si>e <7A e1traction-puri;ication protocol ;or brown macroalgae, +o(ecu(ar 1co(o)y 5otes& 6& ($()($D, 3>ano>a 7J& <ewaard J"& 6ebert P<7 #200B% An ine1pensi>e& automation0;riendl! protocol ;or reco>ering high0@ualit! <7A, +o(ecu(ar 1co(o)y 5otes& .& $$8)(002, 2arakousis A& Tan L& llis <& Ale1iou 6& Hormald PJ #200B% An assessment o; the e;;icienc! o; ;ungal <7A e1traction methods ;or ma1imiCing the detection o; medicall! important ;ungi using P5", Journa( o0 +icrobio(o)ica( +ethods& .5& D8)'8, Liu <& 5oloe 8& Baird "& Pederson J #2000% "apid mini0 preparation o; ;ungal <7A ;or P5", Journa( o0 6(inica( +icrobio(o)y& 27& '/(, Manian 8& 8reeni>asaprasad 8& Mills P" #200(% <7A e1traction method ;or P5" in m!corrhiCal ;ungi, ;etters in -pp(ied +icro= bio(o)y& 22& D0/)D(0, 8uCuki 8& Taketani 6& 2usumoto 2& 2ashiwagi 9 #200B% 6igh0 throughput genot!ping o; ;ilamentous ;ungus -sper)i((us ory,ae based on colon! direct pol!merase chain reaction, Journa( o0 3ioscience and 3ioen)ineerin)& /01& */2)*/', Jogelstein B& Fillespie < #($/$% Preparati>e and anal!tical puri;i0 cation o; <7A ;rom agarose, Proceedin)s o0 the 5ationa( -cade.y o0 /ciences, ?/-& 6.& B(*)B($,
Donald E. Polkinghorne - Narrative Knowing and The Human Sciences (Suny Series in Philosophy of The Social Sciences) - State Univ of New York PR (1988)