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1 The Krean Jurnal f Micrbilgy, Vl. 43, N. 3, September 2007, p 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: , Fax: 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

2 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.

3 212 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 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 Imprved Manual ISOIL kit

4 Vl. 43, N. 3 DNA d m ³ mw S 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

5 214 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 ( ) w w,. š x 1.,, y w (cmmunity DNA). J. Envirn. Sci. Eng. 5, ,,. m w w. 3. w, ½, y S rdna-ardra w ù ù m ü VBNC ³

6 Vl. 43, N. 3 DNA d m ³ mw 215 mw p. w wz 42, y, x » m ³ m r ³. w wz 21, Alexander, M Intrductin t sil micrbilgy. Jhn Wiley & Sns, New Yrk, USA. 6. Amagliani, G., C. Giammarini, E. Omiccili, G. Brandi, and M. Magnani Detectin f Listeria mncytgenes using a cmmercial PCR kit and different DNA extractin methds. Fd Cntrl. 18, Brümmer, I.H.M., A. Felske, and I. Wagner-Döbler Diversity and seasnal variability f β-prtebacteria in bifilms f plluted rivers: analysis by temperature gradient gel electrphresis and clning. Appl. Envirn. Micrbil. 69, Di Pint, A., V.T. Frte, M.C. Guastadisegni, C. Martin, F.P. Schena, and G. Tantill A cmparisn f DNA extractin methds fr fd analysis. Fd Cntrl. 18, Dilly, O., J. Blem, A. Vs, and J.C. Munch Bacterial diversity in agricultural sils during litter decmpsitin. Appl. Envirn. Micrbil. 70, Frtin, N., D. Beaumier, K. Lee, and C.W. Greer Sil washing imprves the recvery f ttal cmmunity DNA frm plluted and high rganic cntent sediments. J. Micrbil. Methds 56, Greene, K New methd fr culturing bacteria. Science 296, Guan, L.L., K.E. Hagen, G.W. Tannck, D.R. Krver, G.M. Fasenk, and G.E. Allisn Detectin and identificatin f Lactbacillus species in crps f brilers f different ages by using PCR-denaturing gradient gel electrphresis and amplified ribsmal DNA restrictin analysis. Appl. Envirn. Micrbil. 69, Ibekwe, A.M., A.C. Kennedy, J.J. Halvrsn, and C.-H. Yang Characterizatin f develping micrbial cmmunities in Munt St. Helens pyrclastic substrate. Sil Bil. Bichem. 39, Insam, H. and K. Haselwandter Metablic qutient f the sil micrflra in relatin t plant successin. Oeclgia. 79, Kaeberlein, T., K. Lewis, and S.S. Epstein Islating Uncultivable micrrganism in pure culture in a simulated natural envirnment. Science 296, Kim, M.J. and J.S. Chun Bacterial cmmunity structure in kimchi, a Krean fermented vegetable fd, as revealed by 16S rrna gene analysis. Int. J. Fd Micrbil. 103, Lagacé, L., M. Pitre, M. Jacques, and D. Ry Identificatin f the bacterial cmmunity f maple sap by using amplified ribsmal DNA (rdna) restrictin analysis and rdna sequencing. Appl. Envirn. Micrbil. 70, Llyd-Jnes, G. and D.W.F. Hunter Cmparisn f rapid DNA extractin methds applied t cntrasting New Zealand sils. Sil Bil. Bichem. 33, Nei, M. and W.H. Li Mathematics mdel fr studying genetic variatin in terms f restrictin endniclease. Prc. Natl. Acad. Sci. USA. 76, Plurde-Owbi, L., D. Seguin, M.-A. Baudin, C. Mste, and B. Rkbi Mlecular characterizatin f Clstridium tetani strains by pulsed-field gel electrphresis and clny PCR. Appl. Envirn. Micrbil. 71, Saitu, N. and M. Nei The neighbr-jining methd: a new methd fr recnstructin phylgenetic trees. Ml. Bil. Evl. 4, Sebat, J.L., F.S. Clwell, and R.L. Crawfrd Metagenmic prfiling: Micrarray analysis f an envirnmental genmic library. Appl. Envir. Micrbil. 69, Sheu, D.S., Y.T. Wang, and C.Y. Lee Rapid detectin f plyhydrxy-alkanate-accumulating bacteria islated frm the envirnment by clny PCR. Micrbilgy 146, Smalla, K., M. Ors-Sichler, A. Milling, H. Heuer, S. Baumgarte, R. Becker, G. Neuber, S. Krpf, A. Ulrich, and C.C. Tebbe Bacterial diversity f sils assessed by DGGE, T-RFLP, and SSCP fingerprints f PCR-amplified 16S rrna gene fragments: D the different methds prvide similar results? J. Micrbil. Methds 69, Stackebrandt, E., W. Liesack, and B.M. Gebel Bacterial diversity in a sil sample frm a subtrpical Australian envirnment as determined by 16S rdna analysis. FASEB J. 7, Takada-Hshin, Y. and N. Matsumt An imprved DNA extractin methd using skim milk frm sils that strngly adsrb DNA. Micrbes Envirn. 19, Tebbe, C.C. and W. Vahjen Interference f humic acids and DNA extracted directly frm sil in detectin and transfrmatin f recmbinant DNA frm bacteria and yeast. Appl. Envirn. Micrbil. 59, Thmsn, J.D., D.G. Higgins, and T.J. Gibsn CLUSTAL W; imprving the sensitivity f prgressive multiple sequence alignment thrugh sequence weighting, psitins specific gap penalties and weight matrix chice. Nucleic Acids Res. 22, Trsvik, V., F.L. Daae, R.A. Sandaa, and L. Ovreas Nvel techniques fr analysing micrbial diversity in natural and perturbed envirnments. J. Bitechnl. 64, Trsvik, V. and L. Ovreas Micrbial phylgeny and diversity in sil. pp In J.D. van Elsas, J.K. Janssn, and J.T. Trevrs (ed.), Mdern Sil Micrbilgy, 2nd. CRC Press, Bca Ratn, FL, USA. 31. Tsai, Y.L. and B.H. Olsn Rapid methd fr direct extractin f DNA frm sil and sediments. Appl. Envirn. Micrbil. 57, Whitehuse, C.A. and H.E. Httel Cmparisn f five cmmercial DNA extractin kits fr the recvery f Francisella tularensis DNA frm spiked sil samples. Ml. Cell. Prbes 21, Wlters, V., W.L. Silver, D.E. Bignell, D.C. Cleman, P. Lavelle, W.H. van der Putten, P. de Ruiter, J. Rusek, D.H. Wall, D.A. Wardle, L. Brussaard, J.M. Dangerfield, V.K. Brwn, K. Giller, D.U. 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7 216 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 , 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.