土壌細菌からの直接DNA抽出法 : 異なるDNA抽出法の比較

全文

(1)k # i # ~ h %DNA~ #lLfZ%:std6 ~ DNA #dlEikCD.k@ ZjiR. *$z. I'. lL'\2'. $$$$. gf?. 2'. Direct Extraction of DNA from Soil : Comparison between Different Methods Kyoung-SOONKANG'),Kiminori ITOH'Ix2, and Masayuki MURABAYASHI2, Synopsis To evaluate the diversity of microorganisms in soil as an environmental index, direct extraction of DNA followed by its analysis is very useful. We compared, in this study, different methods employed in the direct extraction of DNA from the soil. The following method was most effective in DNA yield : that is lysis with a high-salt extraction buffer (1.5 M NaCI) containing 1% hexadecyltrimethylammonium bromide (CTAB) , and proteinase K and extended heating (2h) in 1% SDS solution. Also a method proposed by TOYOTA Co.,Ltd. was effective ; it needed llittle time (lhr) to complete the method extracting process while the other methods needed more than two hours.. DNA Gr)E%@%3EBkf?j!Zi~l%j L L I A e I3 3 TCz%& f2 %?&fii%g $ h T 3 fzO fij ; i$3Bead-Beat i n g :1. h6&#I%f's?@B?& (Cullen b , 1998), El$a> +~~~+fi~ - ; +f B ~ Z + Y I K I H ~ k. j, s a ~ ~ a , &&%#@%g-$-5%?& @ !( B b, 19921, %@% &%@f%izi%L'T@&Lz2sTh 9 ,B~??!il£ia> &$B$@6ikYM@a>%hn?%iz%ET5%?& -92 LL%8$$T8562%2bhEl0 (Clegg 5 , 19971, 8@B% i%?ET%$$-$-a% ?& (Jizhong Zhou b , 1996, U7;- CTAB E 2 @) , @9a>@k+M%Ha>AB$fiJk?& 2 L L , k%rPa>@k L-@%+$a,5-10 {ED SDS '&$&(zd; &M#og%t$fiB$fi&qT 5 7?&tz. k @ b m E a > 2 3u DNA &#Y ,if-a;j -F?&a>&Zrf-WU 16s rDNA 0 $ f i E & a z l i ~ k A % B !(B*$I; ( b 3 9 ;7$ El!!@), 1998, U R+S%?7icB?&iz8+ AB$Fi&f79 LL\5 (gb , T b 3 9 B % ~ ? & k S 5 )f22'fi$&$$T855 2 % 1999) 16s rRNA i 3 & ! % % i ~ ~ ~ ! % l k ~ L @, $ % E e $ i - L T L \ 6 , $$@&< DNA D M t t j f i 4 T S 5 2 . @+*a> 8 b ~%&B29U$B@??$ht&@%+Th 5 19 %< a>*wm~~ma-em,vfir.r%Thi,a. 2 D%*Ti&, TZ&$!&figf2 2'$%% %tz 1 6 s r D N A a>&/k$Bbfi (@Fka?TfCS s f 2 5 fRb3 (Vl-V3)). i%Ba>l%$F t;hLb\66Bbfifi4h STJZHTS 5$%rnhTG$$Th52 % E $ h DNA H$$$@, 9. Z h & % l l H LL&!E3-%@%%%Jf6l??l~-?$Bg L$?zL\< 9 f i W DNA &kt&?&&, a>%%Bfi;WETiba k& (F6, 1995)@k&MD& !+%07%8%$ 7" P t ~ 3 b \ T & ; t ; t ; 2 Z M D k @ . t f Y ~ ~ I ~ Z ? H L \ L* &3&77-> ~@ to f2 b T % M & { $ D B / k %#&&#%-$-5 Tz&D& F$f25++%4%7-%-2 L T , %a&%E< HL\ 2. 5eS b d z T b \ 6 (Woose, 1987), 2 a> k 5 f2, DNA &$j 2.1. gsa)j'L;h $22 L7ZfiF$fit~C2. b-Tfi~DbBlE Lfi>;f:&ttlTS 7'6 k@Y-Y7°1k??%@LL\ 89hTh6 2%%$ L\im.;%td;+ssTdia0 F t~ * g i z $ 3 i - ~ a h 7 ~ ~9 \ 8< 1 DNA@&~S'?&&H~\T ~ DNAM%~ bfl@i5kl$%&ttl-$-a ~Z&~LE@DI\TAM&?&&~&R 77 5 12 C&izi&d7? k 5 iz, tg$$?&d;g &$$T 3-5 2 :t b; 9 k$@pa>@k&M%%a>&&l'$% ;ki&b; 9 iE@k~*&56 2 f i $ 3 J % K f 2 9 T S 7 L z O h5E@DNA&ttl&RL\ikO k@fi>bDNA @ttl';f-. 1. I3CbbIC. k$@fi>btg$$&@2@Ttz DNA ??Mttl L, PCR (polymerase c h a i n r e a c t i o n ) CZ b; 9 @#E L T E. a. <. 0. <. etzqz. z. 1). $g?&@fZ%y Ee$$q@f%?. y240-85-1. @ ~ ~ * f ~ k 79-7 ~. Department of Environmental Physical Chemical, Institute of Environmental Science and Technology, Yokohama National University, Yokohama 240-8 5- 1, Japan. 2). $4%33@7E@S%a CREST, Japan Science and Technology.

(2) 6E, @ @ @ & l t - $ & C ~ @ h T L 3 5 DT, ?a>% %Tb 3 PCR'fis T 3 7'6 L\?z bl, PVPP ( P o l y .. v i n y l p o l y p i r r o l i d o n e ) t Z k 6#%@&?7-> 7z, % L \ 7 PCR ??fTL\, DNA D @ r f r E & ? 7 -xY/LT 8~ @%, L7"zfj$, $gEgg5&*" ,R3s*%?At:S+ %Re. <. fT97co 7 8 a - ; j : ! f / L T t 3 lOObp D%@LfihT 3 f 2 L \a;, 8f;/JJ\%%/j\/L (SPREADEX~EL MINI GEL) T t 3 l b p 3T%%q%T;ti6,. 2. 2.. ssa>fsE!i. f-@-ff27?bB%@ft~i%%. CZ%L\T, L 72". L 7 L \ 6 & 2 , ZHI. 6mfiDSGWfilb l g Dk@&?BB. f-&& 6 O@@DNA &%& DNA #lttj%?& ; 1) k@%$$tZ, @T%% L 7z PVPP (Zoomg/*@ lg) ? ? l l U k 7 $ + V T 4 Y-(Zfili$7z@, 37"CT 1-2 %%74 Y 4 3 - 4 - V s Y 3 - 6 . EL\T 2 0 0 , 1 ~ D 23% sodium dodecyl s u l p h a t e (SDS) ? ? A h , 5O0CT 30 %fikTf&, -80°CTD?@$& g@&?@ 9. BEADS.2)SPB 1 9 y 77- tL 20%SDS, P r o t e i n a s e , $& 0 BEADS3)DNA M ttj 19 y 7 7 bZ 20%SDS,P r o t e i n a s e , Mf&D BEADS, /. 36'@ : k@-!?Y?/b?? (1. Og wet wt) 0 5 { g i% ~ s ~(TE s 7 7 7 - C Z ~ & @ $ e 7 ~ @ )?? L \ ~ E (& 5 3 1 , ~ ~ ~ E D P U U Z I V (LTA EA 777-t~7&7%$@7z@> ??AhE&(5%>@, g l L \ (15000rpm~10 -T*@7Zt$??@q7Fl C S O 4 Y ~ " U / ~ I - / L ~ L \ ~ & E $ @2 ~ ~~ & 9. m. -. I - / ~ ~ , ~ E T D Nb ?A ? f +~6 ,~ / . ; , : *@sf ~ 7 " / ~ (Og 1 .wet. CTRB &. wt) CL ( 1.5M NaC1) , hexadecyltrimethylammonium (UT CTAB k @ < T 6 ) , O . 1 M T r i s HC1 (pH 8 . 0 ) , 0.1M EDTA (pH 8.0) , 0.1M sodium phosphate (pH 8.0) A 9 D DNA # I 8 1 9y 77 -CL A .. fi $$. a%. L\h@&$j$, 7 ' .ST--+! ~ K ? ? A h , 225rpm T & T & 30 ??% Z Y B% ~ SDS 2 L \ h g ? R (65°C) CZ7 2 @?BYC ' -Y 4 Y Y LTglL\ (5000rpmx 10 3 ) L 7 4 + 7 z k B & t r F I B ~ Pa a d v ~ A 2 A h . E A/( a&, * W 7 ? t 3 2 @ ~ r - > - ( r E l C B D 4 Y 7 " ~ / { 1 -. EL, 711-/LMtt:27/L~-/~?kEtZL->T. I L ? ? L ~ ~ L ? ~ E $ @ D N A & Ib/ 2Y$ + 6 ,. DNA ~ t tL ~j Z ,~ 2) V 3 E @ g s i l g z g l L \ ? & t Z L 9 T@#f;/JJ\@f&tg. 2 . 3 . DNA $653 BIO-SPIN 75 7 A ( 4 IcmX 2.5cm). Be??5 o 3) Bead-beating. (Bio Spec. $kg) ? ? H L \ 7 E %. ,@B2?75 4) CTAB ?A : k t (1. 5M NaC1) 2 1%CTAB ??$ DNA j Httlr19y77-&HL\7 i%?RT1' Y 4 = 4 - V 3 2 2 t T 5 (2 %7~7), 5 9 : k@%$$D5--10{gDSDS'@ j@Tt@Bfj$, P u a $ ~ b A # l t t : 2 4 Y 7 ° a 1 ~ 1 /L?kb%iZ& -> T DNA #ltt)&? L k , UTCrt3 3, 4, 5 c G i ? & t r ~ L \ - i C g $L L \ ~ @ @ i???Z-F0 Bead-Beater &$ : kg-!? Y 7 ' / L (1. Og wet wt) 2 Bead-Beater t Z A h 3ml Sodium Phosphate B u f f e r (SPB) , IXSDS, FIED Beads 2hLIkT@, $&6!'J@ ' B ???T3, glb&? fill$ ( 2 8 0 0 ~x 15 %) q% -f:k$$f&iZ.fib-?'< 0.5M EDAT 2 %a) 1/10 E D BE@% 9 9 A (3M) &?Ah,? k k T 20 %?BYE@? $j$ l o , OOOg T 5 %Ba'JglL\ $ @ a t 2 T ~ @ , @ F E E I E & ? R & Tk $~$ ~ f&2FIB~1' ~ 7 ~ u 1 q 1 lL??Ah?kb%$@, 3=9--1/LT&3D7/L3/I.&?@% L T DNA &I/.;, b 2@7z, % IZ bead-beater ? A T 7'6 6 $j!jEi%+$RJ (S'DS) ~ ~ ~ ~ ~ A ~ D ~ ~ . ; b L % k f T 9 7 c o Z Z T , 947'251<.;,77-&T+L <i&+5, 1 ) s 1~ 9 ~~ 7 7 - c z I%SDS, BED. PVPP %% 2 0. 4g (6$ 0. 5cm) Ah$$@* (dH,O) ?? 0. 5m1, 0.25ml bR% t L A h g l L \ L 7 B < (4100rpm, 5 %), #I% L7z DNA A 7 7 " / L (%?I&) 2 1 0 0 , 1~ A h 4100rpm T 5 %VB@E@ 2 !Elf7 5 , %@fi;,$%b6 2 PCR 2?7L\%rfrG&@Z\"P6 Zk. *<, a,. I R J ~ T B7 ~s t- ~F ~~i a 2m, ~~b ;1a z~a %~? ~ &D&fiXIZ 2 PBh6 1El El D#%@f&C~t3, % @@D~~~fi~$@ 7'6E1fibl,Th6, L< DNA &I/.;, b a&Cr& 5 $;, gi@3-5 u I T ' '3 0. a'-. 2.4.. PCR PD%rfiE. PCR TD?$rfrEC31 6 s. Y $Y-A. rp, % t ~ , $Bb$i v i &$fib%. DNA Dglk@mfi. va. &? B m 2 L T. Nested-PCR T f T 5 ( ~ e n e ~ mPCR p System 2400, P e r k i n Elmer), %?, 27f-519r ( & T i 3 Lane tZ L 6%&E$!?lJ DllR) D 7 ' 7 4 -7-&HL\7 V3 $Bmfi&?@F L7z $3, Zbr 27f-109r 2 357f-519r T Vl, V2 $Bb$i?? B-?' L k ; , PCR %#b3 95"C2 5% 1CYCLE, 95OC1 3, 5g0C40 $9, 72"Cl 3 2 30 CYCLE, 72°C 7 %&? 1CYCLE ~ T Tio Q 27f-519r, 357f-519r D 7 ' 5 1'. -7-$R&&b@TC3&$fB@2&D@rfrEfi~T?%!T 3.27f-109r. Tt3KL\$ZBD~flED@rfrEfi~T?%!T (Lane b.. , 3~6. ~F+ LLjY74 - & H ?-D%R&&h@ L \ ~ D 1991) t3&1C Z % L T ; t i 6 ,.

(3) 7°74-7-a> $H&&b* 27f-1091-1 29f2-107CT 29f3-107CT 27f-519r 357f-519r. 2. 5.. ah6 k?BU$h6. % 2& B!l 3rJ 27f : 5'AGAGTTTGATCMTGGCTCAG 109r 1 : 5' ACGYGTTACKCACCCGT 29f 2 : 5' AGAGTTTGATCMTGGCTCAGE 107RcT : 5 ' ACGTGTTACTCACCCGTQ 29 f 3 : 5' AGAGTTTGATCMTGGCTCAGGA 107rCT : 5'ACGTGTTACTCACCCGTCT 27f : 5' AGAGTTTGATCMTGGCTCAG 5 19r : 5' GWATTACCGCGGCKGCTG 357f : 5'CTCCTACGGGAGGCAGCAG 5 19r : 5 'GWATTACCGCGGCKGCTG. *-*-. mx;%al PCR f i ; g b 9 7 ~ g M i a % T , ~ ~ ' U - X Y I L ( 2 %) ( ~ $ L T5 (Mupid, Advance Co., ) DNA a > @ $ g & @ 3 , ~ 7 ~g%@%ti'/~ @, (SPREADEX@ EL MINI GEL) I.ZT%%%@I(Elchrom S c i e n t i f i c Submerged Gel E l e c t r o p h o r e s i s Appratus P/ N2000) ?? LT@E.r"k tr5?t-f6 Z k d4q%T& 6,= %?, -?-4X.-?-3-2 L T M3 -7-3-2 $5L, loading b u f f e r (5x) 1 p 1 CZ PCR &#!I?? 1 1 nnkj.T, s~t7icgw 77-id: 0.75 x TAE, 30 /n\fT 5 . & S E i 2 80V/cm; 17icE@~@r~7 id: 2. *BE%% *Il E *I&$Bi% Y.7-qBf%$BE. &*Illi%*E &*BE@@. i3@@ILf6L\=I2 7266 ( 5 %BaEjfif@9Tg$ZL 7z@~~Yei+B$*6),, 2. 6. @ f5 SYBER Green I n u c l e i c a c i d gel s t a i n (Molecular probes) O E % e % @ & T 10000 %R L T Y I L ~1%rH7t59.3i~7~,. 2. 7.. EZ $@. ~ ~ Y X ~ I L < $ - - Y - AT TO$^%, %%BE 255nm) K Y / ~ e g i %SYBER , Green a>?k%&%jk?? CCD 3 9 7 & f g @ L 7 z F ' Y b~ Y.77(ATTO$*%) iZ k 9 53%L7zo. z 2 129 x I L t z ~ $ $ e A h 6 % ,$YY CTAB %j& f? I f f % h hfc/ C> F'( 193bp). 1. DNA @kb?&l=k Q@l53% ( Z % > , W/n\@%Y)l.. (SPREADEX @E~600). 947h"h. M3 1 L->. :. 2 L->. : CTAB :2 (5g). 3 L-> 4 L->. : @i*53B@Bl$f73fzj&. : fJ%Y>?)l.(Z%). IZPVPP $ A h , SDS T%i;;iLfzj&(5g>. : BEAD-BEATER j& (6g). (1 Og).

(4) 1 t-h@ttj~r~yfix~-w'a T Z ~ g , t ~2 g i;t-gi+ fiiBBT;ti a 7zh, SIBID%~@TI~F$ L~\&ai2 7Tbf2b'Z 2 iz L7z0 % % $ h T 5 7z DNA @ m ? & D p T ,% % ( Z f T q T&Tz%%, PCR iZ k 6@#E-?@i+a>ZT@h7z %?&3-5 iz9b\TT+LL\&Z+%f797zo+&%$& 7z%IlRK k 6 2 %3?&Tf!EHS h 6 +%a>Bfi< g f 2 6 ~ T ZTi2k@%+$a)B&%Ba) Z I g I.r L T k B % f T 3 7 z 0 B I Z iz%%&ZTo. 3. @EBU%% 3. 1. DNA ff8~fS:&DLL$3 BI 1 i Z , %?&5 U9bDk$22fT37z%R%ZTo 7'74 ?--id; 357f-519r %Rb\T@% V2 Ta>$fl B%;k;%Bk, a8 bitrliZ, %?&1 2 2 i2l&a>%?& 2 kdT@$EfixGG < , B@@%DR&fixT$%T ;tia 2 ,@.bh7zo%?&3 i2, f&D%?&TEb h f 2 b>fl'ZE(193bp, 7 VP V J ~ P 9 k,@.bh6) D ,< y p.fiX'ajE3b h , &b\ DNA EJ@+2% Lfz, 2%.. fJj&f? rtg+hhtc/<. CTAB. =/ F' (1 93,194bp). 2. DNA @ ~ ; & kl~%E@/~\?I~DSL\ = (&), S/n\%%YiL(SPREADEX (@ELGOO). M3. :. 94X'7-iJJ. 1 L-=/ : bead-beater. 2 L-> 3 L-> 4 L-9. j&. : b e a d - b e a t e r ( ~ ~ A # h $ ! / S ' ~ - / ~ - 2, 0 % S D S , : bead-beater(~PB/S'71-, :. t-39;& '5. 20%SDS,. y ~ --i?, f. BEADS). ?~f-%',B E A D S ). ~->:cTAB;&. 7'7 4 7-b2 357f-519r &Hb\@b'/i V2 TD$$B %;ki&B7"r0 %{L bead-beating %?&Tid;fC$&iZ k 6 % % % % (Cullen b , 1998) %&q7zi$, g fd: 9 REi%t&j$J (SDS) a>B2@% (Proteinase K) A 9 D / x ~ 7 7 - - 2 H L \ 7 ~ % 3 ? ,1, 2 , 3 1/-7 Ti2853 0 ?S~F$!%fil~ Tz0 Z;lzC2 Cullen %k-&L7"ro 5 1/-Ya3 CTAB ?&C24V - 7 0 b 39?&,1 , 2 , 3 V--j.D Bead-Beating ?&T C"a#bhf2b\lxY F (193, 194bp, 7 1/P 71x3 P - k 1 E . b h 6 ) fixJLbh7z0 z z T, + % ~ fY ~ ~ J L ~ D ~ L \ ~ L ~ L ~ % S f Y 7 ° / l / a > & f i ~ $ f 2 b \t23, b h 6 DNA *;ki DR3iS'filfJ 9 @ b 6 f 22D?Ehitr4;ti60 f,$qT* @Sf ~ 7 ~ 1 ~ g ~ & # f , & ~ % & 6 Z2 & 2 :1 ~ L+ ~ 3 iZ%Tk 5 br, bead-beater :lf, CTAB ?& fz, i;f: l g , 3g 2 b 3 9?&TChO.l g , l g 2 H b \ 7 k. @ L7z0 LA1 L, S f ~ 7 ~ ~ L D & T i " a 3 $ -2 i1<9 C3iZ 2 X/ y @ b b f2is13fzO fz?? L , *%Sf ~ 9 ' ll/gfi:&b\k+f2b\!l*k kd, 4+b$L9lx2 p.(2 3$ < f2 37z0 Q 4 i-h 16s Y $ Y --A DNA a>@fCfJjb$I (V 1) % E i ~ 9 '4 7~ ~ ~ B ~ ~ &?z%3?T%;;5, ~ b \ k % H & & b - @27f-109rl i2 ( e $ $ B ) , 29f2-107CT ($I&$flE), 29f3-107CT (?7AP&k$flE) T 6 6 0 %@$i?&%i~42%l3@, $I&$$E,Y-7AEt&$FllBi2%f26$$B%fi@% \ LTk S ~ ~ , O%@$i?iki~k667A/%1.&$fl@2 Y~AP$$ t&$i3@iZ$fT6l<YFDl<P-Yi&iZ2 X/ym c ~ h - 7L ~A ~~L0 , ~ 3 ~%~ ; t i 6 3 % f l E i ~ V - 7 2 k 4 5 k CTAB ?&iZf&D%?&Ti2R h h fJb\{cEDlx7 F (114bp) fixBb h , ft?ID%?& 1 9 I ~ % $ $ ' $ D D N A B ~ YT~2~7~z@ k l <R . b h 9 ,.

(5) Sfii6 k i S - c f > ?)Lf. 3. 4. M3 1 L-> 3 L->. :. 5 L-> 7 L->. :. $3 DNA. IZ 6$&5k%/n\6DZZ. (SPREADEX @EL600). V4Z.7-33 2 L-> : BEAD-BEATER l g 4 L-=/ : BEAD-BEATER 3g. : BEAD-BEATER l g. : BEAD-BEATER+PVPP l g. l-39;2. l-39% 0.1g. 6 L->. :. : CATBjS l g. 8 L->. : C A T B j S 3g. 1g. 3'7 4 ? - D @ & ~ ; ~ - ~ ~ I Z & Q( a~) ,ESPREADEX D ' ~ @EL300 ' ~ ~ ~ L. 1 L->. : 27f -109rl. 2 L->. : 29f2-107rl. CT CT. 3 L->. : 29f3-107rl. 7 L->. : 27f -109rl. 8 L-9. : 29f2-107rl C T. 9 L->. : 29f3-109rl. I. 4 L-> BEAD-BEATER ;%. : 27f -109rl 5 L-=/ : 29f2-107r C T 6 L-> : 29f3-107r C T. CT. rhcaz%a>mw -e-g~~f-~ 7 ~ 9 7 CTAB E T-ta+$t~&L\~@ 2 DNA a > B l l ~ d 4 q % T h 6 ~ 2 i24&a>fi?&. ( a ) , &/n\%i%Yi'lL. 2 b k 4 7 ? B E E 4 F ??B?R. ( 6 5 0 ~ )TE6B7779 TL\EIt 2 tz .L6 2 , ~ \ b 6,3 2 iC%-?%a> DNA @186?~7, '%i%?El$E,DNA dr/y. b a g , D N A d r / . ; r ba>E&Zk&7z0. t ~.

(6) BEAD-BEATER $2. DNA ~ ~ k l ~ r n 7 ?@%iE@ (83rn7) D N A d V y ba>& D N A d V y ba>&. 2. bgP?&. 3 8 3r~7F. 1. 40°C (5 /n\). 0 @LX%&. CTAB ?&. %Xi.(5 3). 65°C (2 8%r~7). 0. 0. ?8&. $865. 3) DNA @ j g C C PVPP h &ML\kA 7 A @ j ~ f i x g F $ 3 . 2 . PVPP $HL\Ta) DNA %9$ T h 9 Tco k?!(O&%?&ThbLkl L7'r DNA i3S 2 kZ%LTzd; 5 trjE&T;ti 9. &&a>@t~@&$X/TL\6?z& #&fixiZ\ZT;ti6,, L \ < ~ f i h ~ % ? & & % L ? z % & R ,3IR*rn Clegg. C.D., K.Ritz and B.S. Griffiths 1997. PVPP kr dc 6 A 7~ # % g f i ' R $ $;'ro ~2 h t~.~ i C - h , Direct extraction of microbial community DNA z ~ > % ? ~ ~ z T L \ TL E< FS d T E Cullen b [ Z from humified upland soils, Letters in d; 5 2 PVPP A 7A%@&--ElfT->TzfS,Sephadex Applied Microbiology ,25 : 30-33. G-75 C L % ~ L T Z A ~ A @ ~1!E&f & Tj Z 2TfCullen.D. W. and P. R. Hirsch 1998. Simple and 3 T h 6 2 L \ 5 z 2 Tffi', %E%L?c%R, -El Rapid Method for Direct Extraction of a> PVPP 27 7A%%? i3i2 2 X/ Pa>h$$E@ 2 ,Fib Microbial DNA from Soil for PCR, Soil d?J5?8&iS;%&T3 Tzfix PCR Ta>@$gfilRkfJ?iSh Biol. Biochem. , 30 : 983-993. 9TzTz&, + & a > @ E @ @ E D % % f i ~ ~ 7 6~2h Jizhong.Z., Mary. A. B. and James M. Tiedje 1996. ,E,bd?JTzo5 Tz, Sephadex G-75 I.L*@ Lk7S 7. <. A%%%&L\%%fil436hf2iSb~?z?z&, PCR a> @@%SLf2fJ'b, i$%%&f6 PVPP ? J y A % % & 3 Eli31 b 5 El779 to. DNA Recovery from Soils of Diverse Composition, Appl. Environ. Mocrobiol. , 316-322. Lane. D. J. 1991. Nucleic Acid Techniques in Bacterial Systematics, 115-175. 4. % ;fi e Woose. C.R. 1987. Microbiol. Rev, 51 : 221-271. 1) ENA hbLkl%?&a>5 %, %iZ$$%+%2 I%@ $T ?%lf~fJT 6f i b 9 Tz BEAD-BEATER &, b 3 9 ?&, M+$+?fi, IhT%S, RL4%k ( b 3 9 El @@I , 1998. ;iPrB%zT, % M q 10-66599. CTAB ?&@hk@L?c%R, I&a>%?&TiC-hE b h f2Lj DNA I < Y F ( 7 k3'5'1x3' 9 - 2 , F , b h S El@?, ~J\%AP, iF@G$Z,+$fiEfT 1999, 3% 66 EE@ZIL%& p. 91. a ) f i : q + b i m j w . DNA BQ+T~~CTAB ?% fix%.% &Ah->Tzo DNA #ILkl@~~~@~n'l%TiC-h q 6 W 1995. S%&%&fjfj% 18 3,1-6 7 b1 @/37T%7T6, b 3 9?&fi:!$fZhTL\Tzo %Z&4k, 2) $@%a> El & T h 6 * k @ % ! & @ 3 6 & 8 jR%- %, EE%$&,ST?@& 1992. % # ~ ~ ~ # ! ?. 2 T%i&%~a>&&+%&k@T6@R. +@Z%?&, k@%!+@R%2#G, ??$Biz. Cr&w-ct~&Ba>-tf~7"1~Bbr-f&~~fJ?L\ z 163-164. 2 ilx*i319 Tzo.

(7)