The Krean Jurnal f Micrbilgy, Vl. 43, N. 3, September 2007, p. 210-216 Cpyright 2007, The Micrbilgical Sciety f Krea DNA m d ü ³ mw Áw Áy * w k w w manual ISOIL kit w m d m l w DNA 16S rdna PCR s clningwš clne w ARDRA cluster ww, manual w 136 clnes 45 ARDRA cluster, ISOIL kit w 76 clnes 44 ARDRA cluster. ƒ clne cluster l t clne w 16S rdna» w, ISOIL kit 44 t clne α-, β-, γ-, δ-prtebacteria, Acidbacteria Actinbacteria 3 phylum m y, manual w 45 t clne α-, β-, γ-, δ-prtebacteria, Acidbacteria, Bacterides, Verrucmicrbia, Planctmycetes, š Gemmatmnadetes 6 phylum w m. l manual w DNA mw w ww ƒ w m w x. wr, ISOIL kit w clne 40%ƒ α-prtebacteria m w, 30%ƒ γ-prtebacteria m w m y, manual w clne 41%ƒ Acidbacteria m w š -prtebacteria (28%)ƒ m sw mw p ùkü DNA m ³ mw p w ùkùš. Key wrds ý DNA extractin, humus frest sil, ISOIL kit, manual methd, phylgenetic diversity m 1 g 10 9 ~10 ³ w x 10 ü ƒ w 1% w (1, 3, 4, 11, 15). m kw mw š m y l w DNA š w m w» q w» w FISH, DGGE, PCR-SSCP, PCR-T-RFLP š ARDRA w w» w m w w š (7, 12, 13, 17, 24). w» mw x m ³ 300,000~1,000,000 š (16).» w swwš m mw w ww» w DNA PCR s v x Ÿ w š DNA (rapid methd) ww m ü PCR w y PCR s œ ƒ w DNA w» w ƒ š(1, 18, 26, 27, 31, 32), w DNA r y w Kit w *T whm crrespndence shuld be addressed. Tel: 82-42-829-7598, Fax: 82-42-829-7599 E-mail: kswhang@mkwn.ac.kr kw ƒ y w w š. DNA mw š kw w w w y ü Ásƒw (6, 8). m» ù ù ƒ ù» mw wƒ w d(humus layer) x w.» w w m,,,» m ü yw w ³ x w (2, 5, 13, 14, 33). x Ÿ w š DNA (Rapid ) w m ü z w manual w DNA wš,» Kit DNA ƒ rwš y ü z w ISOIL kit w m d m l ƒƒ w DNA ³ mw mw. m œ w ù (Quercus acutissima Carruth) w. 210
Vl. 43, N. 3 DNA d m ³ mw 211 ù» ù ù ƒ ù wƒ w d(humus layer) x w. ù ù d 15~20 cm wd d l w. manual w ttal DNA m d w l ttal DNA Tsai w sdium ddecyl sulfate (SDS) ƒwš freezing thawing w s w Rapid (10, 18, 31) w cell lysis z, CTAB ƒw w manual ttal DNA w. m 5 g 120 mm phsphate buffer (ph 8.0) 10 ml š 37 C w» 12,000 g 10 xkw z, 12,000 g 10 w w.» m 3z w m ü humic acid PCR s w w. m slutin I (150 mm NaCl, 100 mm EDTA, D.W. 100 ml, lyszyme 1 g; ph 8.0) 8 ml ƒwš 37 C 2 k z, slutin II (100 mm NaCl, 500 mm Tris-HCl, D.W. 100 ml, SDS 10 g; ph 8.0) 8 ml ƒw -70 C deep freezer þ 65 C 3z w z, 5,600 g 10 w. z 5M NaCl 2.7 µl 10% CTAB 2.1 µl ƒwš 12,000 g 10 wš w 1.6 M NaCl sw 13% PEG ƒw DNA pellet. DNA pellet w z 750 µl d.w ƒw 37 C š 10 M NH 4 OAc 190 µl, 1.5 ml ethanl š 750 µl isprpanl ƒw DNA wš 70% ethanl 1 ml w z œ (Micr Vac MV-100, TOMY)w. 50 µl D.W. ƒw DNA š» (Mupid-21, Gel dcumentatin system, Bi-Rad) mw y w. DNA Spectrphtmetr (UV- 1650PC, SHIMAZU) w d w Ÿ A 260/ A 280 w. ISOIL Kit w DNA Takada bead beating (26) DNA skim milkƒ ƒ m DNA z ƒ k w š x ISOIL kit w d w l ttal DNA w. m 0.5 g ISOIL kit (Nippn Kit c., Japan) lysis slutin BB 950 µl ƒw bead beater (Fast prep. Inc., Funakshj. Japan) 6 m/sec, 45 wš, 60 C 1 k z 10,400 g 1 w. purificatin slutin 400 µl ƒwš z 600 µl chlrfrm ƒ wš 15 vrtexw z 10,400 g 15 w. precipitatin slutin 800 µl ƒw yww z 12,000 g 15 w w. e washing slutin 1 ml z 12,000 g 10 w š wš 70% ethanl 1 ml ethachinmate 2 µl ƒw 12,000 g w z DNA pellet. DNA pellet œ (Micr Vac MV- 100, TOMY)wš 100 µl TE buffer ƒw» (Mupid-21, Gel dcumentatin system, Bi-Rad) mw DNA y w, Spectrphtmetr (UV-1650PC, SHIMAZU) y wš Ÿ A 260/ A 280 w d w. 16S rdna PCR s 16S rdna sw» w E. cli 16S rdna cnserved sequence» w 27F (5'-AGAGTTTGATCCT GGCTCAG-3') primer 1492R (5'-AAGGAGGTGATCCAGCC GCA-3') primer w (3). PCR 1 PCR buffer, 0.2 mm dntps, 0.2 µm primer, 2.5 U Taq DNA plymerase (Slgent c., Krea), template 10~50ng w. v 50 µl w s»(geneampr PCR System 9700, Applied Bisystems) w 94 C 5 w 94 C denaturatin 1, 58 C annealing 30, 72 C extensin 1 30z wš 72 C 10 final extensin g. PCR s 1% agarse gel» (Mupid-21, Gel Dcumentatin system, Bi-Rad)w s y w (20). s 16S rdna ethanl precipitatin w z, 30 µl D.W. ƒw -20 C w. 16S rdna clning s 16S rdna clning pgem-t Easy Vectr Systems (Prmega, USA) w w (7, 22). DNA 50 ng, 1 buffer, T Easy Vectr 50 ng, T4 DNA Ligase 1 µl D.W. 10 µl 4 C 12 w ligatinw. 1.5 ml micrtube cmpetent cell 50 µl(10 8 cell/ml) ligatin 5µl yww z 1 ewš 42æ 90 heat shckwš, 2 ew. LB 450 µl ƒw 37 C 144 g 1 k w z, X-gal (20 mg/ml) 1 ml, ITPG (20 mg/ml) 100 µl, š ampicillin (20 mg/ml) 1 ml sw LB w w, 37 C 24 w. LB plate white clny w clne library z T7 (5'-TAATACGA CTCACTATAGGG-3') primer SP6 (5'-TATTTAGGTGACACT ATAG-3') primer w clny PCR ww (20, 23). clny PCR w 1 PCR buffer, 0.2 mm dntps, 0.2 µm primer, 2.5 U Taq DNA plymerase (Slgent c., Krea) D.W. 50 µl 0.2 ml PCR tube š yw w z 95 C 5 w 94 C denaturatin 30, 55 C annealing 30, 72 C extensin 1 30z w š 72 C 7 final extensin PCR (GeneAmpR PCR System 9700, Applied Bisystems) w.
212 Hee-Seng Sn et al. Kr. J. Micrbil Amplified Ribsmal DNA Restrictin Analysis (ARDRA) clne 16S rdna PCR s w wz HaeIII (Prmega, USA) AluI (TAKARA, Japan) ƒƒ w ùkù band w (3, 19). PCR prduct 1 µg, 10 buffer 2 µl, enzyme (500 unit) 1 µl, D.W. 20 µl tube z 37 C 4 g. wz w 4% agarse gel (1 TAE buffer; 40 mm Trisacetate, 1 mm EDTA) w 100 V, 300 ma 1 30» w z ethidium brmide (EtBr) 30 w UV (Gel dcumentatin system, Bi-Rad)w wz y w. y band pattern Gel Cmpar II prgram (versin 4.0; Applied Maths, Belgium) w band r(restrictin fragment; RF) w. ƒ band r Dice's similarity cefficient (S) wš, dendrgram UPGMA (unweighted pair grup methd using arithmetic average) w (18, 21). S=2 n xy /n x +n y n xy : w œm DNA r n x /n Y : w ƒƒ w DNA r m w mw ³ ƒ ü ³ (Diversity Index) Margalef w Shannn-Weaver funtin (H') œ w w š(25), ³ (Evenness Index) Pielv w w w (9). 16S rdna» m w 16S rdna x ABI PRISM BigDye Terminatr Cycle Sequencing Ready Reactin Kit (Applied Bisystems) w» w. Sequencing PCR BigDye 1.3 µl, T7 primer 1 µl, 16S rdna sample (100 ng) 1 µl, 5 buffer 3.4 µl D.W. 13.3 µl yww z cycle sequencing w. PCR 100% ethanl 50 µl 3 M sdium acetate (ph 5.2) 2 µl ƒw z 12,000 g 20 wš, 250 µl 70% ethanl w k z HiDi Frmamide 20 µl ƒw 95 C 2 denaturatinw z þƒ jš ABI PRISM 310 Genetic Analyser (Applied Bisystems) w 16S rdna (350~600 bp)» w š, RDP II (Ribsmal Database Prject II) database Sequence Match prgram w w. ƒ» alignment clustal X prgram w w š m w (28) w (21). š m l DNA 16S rdna PCR s m l DNA w DNA wì (humic acid) PCR w w» w skim milk w, gel elutin filteratin mw DNA Kit w w w š (27, 29). kw š Rapid Rapid PCR w z w» w CTAB (cetyltrimethylammnium brmide) ƒw w manual š bead beating mw m ü s ³ š skim milk š DNA ISOIL kit w m d m l DNA wš PCR s sƒw. m Rapid w DNA ( Ÿ ; O.D 260 ) 105 ng/µl (A 260 )ƒ 0.89 û,» ƒw manual w DNA w, DNA 110 ng/µl m Rapid j ƒ ù (A 260 )ƒ 1.13 d. wr, ISOIL kit w DNA w, (A 260 )ƒ 1.79 w. ù DNA ƒ 98 ng/µl manual w û ùkû (Table 1).» w DNA 50 ng/µl w 16S rdna PCR s w. Rapid w DNA 16S rdna PCR s ù, manual ISOIL kit w DNA x w œ 16S rdna PCR s (Table 1). l 0.5 g m w ISOIL kit w 5g m w DNA w rapid manual DNAƒ. ù m š w, ƒ û DNA ISOIL kit w q. DNA y w PCR w z» Rapid w DNA m ü w m ³ w w š q. Table 1. Cmparisn f DNA yield, quality and PCR amplificatin f humus frest sil by using different extractin methds Methd DNA extractin DNA yield (µl/ml) Quality (A 260 ) Amplificatin t PCR Rapid 105 0.786 - Imprved Manual 110 0.984 + ISOIL kit 98 1.109 +
Vl. 43, N. 3 DNA d m ³ mw 213 16S rdna Clning ARDRA pattern DNA mw p w ùkù š kw w w w y ü Ásƒw (18). ù m d ü ³ mw sƒw» w» manual ISOIL kit w ƒƒ DNA 16S rdna PCR s pgem-t easy vectr clningwš clne w ARDRA (amplified rdna restrictin analysis), HaeIII AluI wz ƒƒ w ùkù DNA fragment m ³ w w ù ƒ d ü ³ mw mw. d m l DNA 16S rdna PCR s clningw manual w DNA 136 clnes š ISOIL kit w w DNA 76 clnes w. clnes w wz HaeIII AluI ƒƒ ƒw ùkù 16S DNA r (restrictin fragment; RF) Gel Cmpar II prgram (versin 4.0; Applied Maths, Belgium) sftware w ƒ clne wš UPGMA w Matrix w y w. manual w 136 clnes genus ƒ ƒ w 70% 45 ARDRA cluster (17) ISOIL kit w 76 clnes 70% 44 ARDRA cluster y (Fig. 1). d m ü ³ mw ù d l DNA w ƒ clne cluster l t clne w 16S rdna» wš, RDP II (Ribsmal Database Prject II) database Sequence Match prgram w ww. ISOIL kit w DNA 44 16S rdna-ardra clne cluster l t clne 16S rdna» y w α-, β-, γ-, δ-prtebacteria, Acidbacteria Actinbacteria phylum 3 m y. ƒ 16S rdna-ardra clne cluster w clne Caulbacterales (4 clnes), Rhizbiales (20 clnes) š unclassified alphaprtebacteria sww α-prtebacteria m Burkhlderiales (6 clnes), unclassified betaprtebacteria (2 clnes) sww β-prtebacteria m, Xathnnadales (6 clnes), unclassified gammaprtebacteria (15 clnes) sw w γ-prtebacteria m, unclassified deltaprtebacteria m (2 clnes), Acidbacteriaceae m (12 clnes), Micrbacteriaceae m (1 clne) š uncultured Actinbacteria m (2 clnes) y (Fig. 3). manual w ƒ 16S rdna-ardra clne Fig. 1. Dendrgram based n UPGMA clustering (A) and distributin f 16S rdna ARDRA clusters (B) f clnes frm humus frest sil with the restrictin endnucleases AluI and HaeIII. Extractin DNA by using imprved manual methd(left), Extractin DNA by using ISOIL kit (right). cluster l 45 t clne 16S rdna» w α-, β-, γ-, δ-prtebacteria, Acidbacteria, Bacterides, Verrucmicrbia, Planctmycetes, Gemmatmnadetes phylum 6 w m y. ƒ 16S rdna-ardra
214 Hee-Seng Sn et al. Kr. J. Micrbil Fig. 3. Cmparisn f humus frest sil bacterial diversity index and evenness index t each DNA extractin methd. ý ; ISOIL kit, þ ; imprved manual methd. Fig. 2. Cmparisn f humus frest sil bacterial clnes belnging t each phylum. ISOIL kit (A) and imprved manual methd (B). 1, Caulbacterales; 2, Rhdspirillales; 3, Rhizbiales; 4, unclassified alphaprtebacteria; 5, Burkhlderiales; 6, unclassified betaprtebacteria; 7, Xanthmnadales; 8, unclassified gammaprtebacteria; 9, Myxcccales; 10, unclassified Delta prtebacteria; 11, Acidbacteriales; 12, Sphingbacteriales; 13, Actinbactales; 14, Verrucmicrbiales; 15, Planctmycetales; 16, Gemmatimnadales. clne cluster w clne unclassified Rhdspirillales (12 clnes), Rhizbiales (22 clnes) š unclassified alphaprtebacteria (4 clnes) sww α-prtebacteria m Bulkhlderiales (5 clnes) š unclassified betaprte-bacteria (17 clnes) sww β-prtebacteria m, unclassified gammaprtebacteria m (1 clne), Myxcccales (1 clne) sww δ-prtebacteria m, Acidbacteriaceae (56 clnes) sww Acidbacteria m, Crentrichaceae (1 clne) Sphingbacteriaceae (5 clnes) sww Bacterides m, uncultured Verrucmicrbia bacterium (3 clnes) sww Verrucmicrbia m, unclassified Planctmycetaceae (7 clnes) sww Planctmycetes m š Gemmatimnadaceae (2 clnes) sww Gemmatimnadetes m w m y (Fig. 2). m d ü sw ³ mw w» w ƒ ARDRA ³ y w. ISOIL kit w, ƒ 4.85 ³ 0.87 ùkû. w, manual w, 5.04, ³ 0.92 y (Fig. 3). l manual w DNA w ƒ ISOIL kit w t w ³ y w x.» ISOIL kit w ƒ clne 40%ƒ α-prtebacteria m w, 30%ƒ γ-prtebacteria m w m ùkûš, Acidbacteria (15.8%), β-prtebacteria (10.4%), Actinbacteria (3.9%) δ- Prtebacteria (2.6%) sw mw p ùk ü. wr manual w DNA w clne 41.3%ƒ Acidbacteria m w, 27.9% ƒ α-prtebacteria m w m ùkûš, β-prtebacteria (16.2%), Planctmycetes (5.1%), Bacterides (4.4 %), Verrucmicrbia (2.2%), Gemmatmnadetes (1.5%), γ- Prtebacteria (0.7%), δ-prtebacteria (0.7%) sw mw p ùkü DNA w m ³ mw p w ùkùš. kw x rwš h w DNA w š m ü w w DNA Kit w m ³ mw w wwš ù, x m ³ mw w w w m p DNA š w mw mƒ v w š q. m kw» y» w. 2007 21 (200503 0134384) w w,. š x 1.,,. 2003. y w (cmmunity DNA). J. Envirn. Sci. Eng. 5, 17-19. 2.,,. m w 2002. w. 3. w, ½, y. 2005. 16S rdna-ardra w ù ù m ü VBNC ³
Vl. 43, N. 3 DNA d m ³ mw 215 mw p. w wz 42, 116-124. 4. y, x. 1995.» m ³ m r ³. w wz 21, 319-324. 5. Alexander, M. 1985. Intrductin t sil micrbilgy. Jhn Wiley & Sns, New Yrk, USA. 6. Amagliani, G., C. Giammarini, E. Omiccili, G. Brandi, and M. Magnani. 2007. Detectin f Listeria mncytgenes using a cmmercial PCR kit and different DNA extractin methds. Fd Cntrl. 18, 1137-1142. 7. Brümmer, I.H.M., A. Felske, and I. Wagner-Döbler. 2003. Diversity and seasnal variability f β-prtebacteria in bifilms f plluted rivers: analysis by temperature gradient gel electrphresis and clning. Appl. Envirn. Micrbil. 69, 4463-4473. 8. Di Pint, A., V.T. Frte, M.C. Guastadisegni, C. Martin, F.P. Schena, and G. Tantill. 2007. A cmparisn f DNA extractin methds fr fd analysis. Fd Cntrl. 18, 76-80. 9. Dilly, O., J. Blem, A. Vs, and J.C. Munch. 2004. Bacterial diversity in agricultural sils during litter decmpsitin. Appl. Envirn. Micrbil. 70, 468-474. 10. Frtin, N., D. Beaumier, K. Lee, and C.W. Greer. 2004. 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216 Hee-Seng Sn et al. Kr. J. Micrbil ABSTRACT : Cmparisn f the Phylgenetic Diversity f Humus Frest Sil Bacterial Ppulatins via Different Direct DNA Extractin Methds Hee-Seng Sn, Sng-Ih Han, and Kyung-Sk Whang* (Institute f Micrbial Eclgy & Resurces and Department f Micrbilgy, Mkwn University, Daejen 302-318, Krea) The principal bjective f this study was t analyze 16S rdna-ardra f the humus frest sil via an imprved manual methd and an ISOIL kit n the basis f the UPGMA clustering f the 16S rdna cmbined prfile, 44 ARDRA clusters f 76 clnes via the ISOIL kit methd and 45 ARDRA clusters f 136 clnes via the imprved manual methd. On the basis f the 16S rdna sequences, 44 clnes frm the ARDRA clusters by the ISOIL kit were classified int 3 phyla : α-, β-, γ-, δ-prtebacteria, Acidbacteria and Actinbacteria. Using the imprved manual methd, the specimens were classified int 6 phyla : the α-, β-, γ-, δ-prtebacteria, Acidbacteria, Bacterides, Verrucmicrbia, Planctmycetes and Gemmatmnadetes. As a result, the mdified manual methd indicated greater phylgenetic diversity than was detected by the ISOIL kit. Apprximately 40 percent f the ttal clnes were identified as α-prtebacteria and 30 percent f the ttal clnes were γ-prtebacteria and assigned t dminant phylgenetic grups using the ISOIL kit. Using the mdified manual methd, 41 percent f the ttal clnes were identified as Acidbacteria and 28 percent f ttal clnes were identified as α-prtebacteria and assigned t dminant phylgenetic grups.