w w m y wz Vol. 14(3), p. 57~67, 2009 œ 토양및퇴적토환경시료로부터 DNA 추출하는방법에대한고찰 Á Á y* w m y œw A Review on the Current Methods for Extracting DNA from Soil and Sediment Environmental Samples Keunje YooÁJaejin LeeÁJoonhong Park* School of Civil and Environmental Engineering, Yonsei University, Seoul, Korea ABSTRACT In soil and sediment environment, microorganisms play major roles in biochemical cycles of ecological significant elements. Because of its ecological significance, microbial diversity and community structure information are useful as indexes for assessing the quality of subsurface ecological environment and bioremediation. To achieve more accurate assessment, it is requested to gain sufficient yield and purity of DNA extracted from various soil and sediment samples. Although there have been a large number of basic researches regarding soil and sediment DNA extraction methods, little guideline information is given in literature when choosing optimal DNA extraction methods for various purposes such as environmental ecology impact assessment and bioremediation capability evaluation. In this study, we performed a thorough literature review to compare the characteristics of the current DNA extraction methods from soil and sediment samples, and discussed about considerations when selecting and applying DNA extraction methods for environmental impact assessment and bioremediation capability evaluation. This review suggested that one approach is not enough to gain the suitable quantity and yield of DNA for assessing microbial diversity, community structure and population dynamics, and that a careful attention has to be paid for selecting an optimal method for individual environmental purpose. Keywords : Soil DNA extraction, Direct lysis extraction, Indirect lysis extraction, Soil microorganisms m m n m y w y w w š. w w m k m n m ky sƒw t w. yw w w m n m l v w DNA y w w. ¾ m n m DNA w w w» ƒ, y k wsƒ w» sƒ DNA w e w. x mw w m n m ü DNA p Á wš, x š DNA m n m y sƒù w m sƒ y w š w w» w w š w. mw wù p, y w» DNA w, m n m ww k w. : m DNA, w, w, m 1. *Corresponding author : parkj@yonsei.ac.kr š : 2009. 5. 8 : 2009. 5. 20 : 2009. 6. 24 m : 2009. 8. 31 ¾ 57
58 Á Á y m k m» w yw w p x y ¼ w š w (Sylvia et al., 2005). w m n ü š» ww m w. m m» 1% wš wš, z w» w» w w m n ü yw wš. m wá mw y y y» š j» m n m y sƒwš w t x š (, 2000). x w x s sww wš w (Martin-Laurent et al., 2001). x m ü m t l s wü» w m,»» m l s. x p w» w w ù w yw ww» w ³ k w zw (Lindahl and Bakken, 1995). ù x y w. k w w w y w 1% x xþ (Amann et al., 1995). w y w DNA/DNA hybridization, w kinetics š 16S rdna s w (Torsvik et al., 1990; Von Wintzingerode et al., 1997). y w l DNA w w, t w DNA z w (Wilson et al., 1997). w DNA š w w w w. x ¾ m n m DNA w» w» w y š t y. m n m ü ü, x» ù y kit wš x. w y m n m y DNA w Fig. 1. Schemes of two different approaches to nucleic acid extraction from soil sample. w w w e ù ƒ x w ƒ». m n m y ü k» w w x š w DNA w x wš p w m w» m DNA w ù š w w w š w. m n m l w w Fig. 1 j 2ƒ. w in situ s wwš m l w w z, w w (Ogram et al., 1987). w, m s wš w w z w wš, w (Holben et al., 1988; Courtois et al., 2001). ƒ DNA, DNA ƒƒ š (Roose-Amsaleg et al., 2001). m n m ü k tw z DNA w» w w w w Á w š, x mw ƒ p w š w. 2. w in situ s w ù 10. w in situ w š ƒ w w ü š DNA œw. Ogram et al.(1987)
m n m y l DNA w w š 59 Table 1. Characteristics of cell lysis methods Cell Lysis method yw z Class» 2ƒ w (Fig. 1). s sü w w. w w s, ü s e, š m y l w. w m l wš, w w w w. 2.1. s w x s w( ) Table 1 (i), (ii) yw, š (iii) z w, ƒ w š (Hurt et al., 2001). m q w m ü ¾ š s sww s w w. ƒ -w (freezing-thawing), - (freezing-boiling) bead w ³ y(bead-mill homogenization) (Steffan et al., 1988; More et al., 1994; Degrange et al., 1995; Miller et al., 1999; Maarit Niemi et al., 2001; Miller et al., 2001). Mortar mill Characteristics -w (freezing-thawing) ƒ w. - (freezing-boiling) ƒ w. bead w ³ y (bead-mill homogenization) ƒ w. Bead-beating, beating wš (extraction buffer) DNA ƒw, DNAƒ ƒ w yw w. w ù EDTA, Chelex 100, w s w j Tris bufferù sodium phosphate buffer k p (chelating agent) sodium dodecyl sulfate (SDS) w EDTA ƒ k w ƒ y ù, w û Cetyltrimethyl-ammonium boromide (humic compound). (CTAB) x ù, s qr w x w Polyvinylpolypyrrolidone (PVPP) (lysozyme treatment) achromopeptidase Proteinase K (humic compound). CTAB w DNA w PVPP s w z, w spin column w z z w ƒ. ƒ w DNA ƒ k. ƒ w Gram-positive ³ w j wƒ grinding, y, q, j q š (Picard et al., 1992; Tebbe et al., 1993; Zhou et al., 1996; Porteous et al., 1997; Orsini et al., 2001). Frostegard et al.(1999)» m w z w g w. xk(vegetative form), s s z ƒw DNA w w» w (Liesack et al., 1991; Simonet et al., 1991; Miller et al., 1999). w DNAƒ š DNAƒ v w (Fosmidù Cosmid metagenomic cloning š DNAƒ ù operon x w ) w ƒ. yw w» wš w» w. ƒ w s w j sodium dodecyl sulfate(sds) w. p Table 1». w Chelex 100 w ƒ DNA û z ü y w (Miller et al., 1999).
60 Á Á y Table 2. Nucleic acid purification methods Purification method ƒ» (agarose gel electrophoresis) Sephadex G200 G150, Sepharose 2B, 4B, 6B Biogel P100 P200»k (Promega, Amicon, Quigen ) w z yw» w w. Selenska Klingmüller (1991)ƒ šw w DNA 70 SDS-sodium phosphate buffer ü m xk» š. w s³ 25 kb j»ƒ j w. w» Cetyltrimethylammonium boromide(ctab) polyvinylpolypyrrolidone (PVPP) ƒ w š (Von Wintzingerode et al., 1997; Kresk et al., 1999; Nalin et al., 1999). x guanidine isothiocyanate m l mrna w m l DNA z x (Tsai et al., 1991; Miller et al., 1999; Orsini et al., 2001; Marrit Niemi et al., 2001). z w w (Bruce et al., 1992). (lysozyme treatment) s t ww ƒ (Rochelle et al., 1992; Tebbe et al., 1993). s wƒ Gram-positive ³ w w achromopeptidase w» wš, w w ƒ w proteinase K w.(table 1) z w, yw w w DNA ƒ wù, š g DNA w. 2.2. w m w wš w ƒ (Zhou et al., 1996). m (humic acid) DNA restriction enzyme digestion polymerase chain reaction(pcr) Characteristics DNA s» w l PVP(Polyvinylpyrrolidone) w» l DNA w Sepharose l DNA w Microspin Sepharcryl S-300 S-400 columns (Pharmacia Biotech)» DNA w» 97%ƒ y j m v (ion-exchange chromatography) mw ƒ ƒ w wwš hybridization signal û membrane hybridization x k (Tebbe et al., 1993; Alm et al., 2000). ü phenolic group amide ù y DNA œ w mw i (quinone) x wš, w k (Young et al., 1993). s w z DNA w ethanol, isopropanol polyethylene glycol w e» (phenol ù chloroform ) l (Steffan et al., 1988). w» (crude extracts) DNA, yw» w w (Ogram et al., 1987). w hydroxyapatite column l œ DNAƒ š, m n l DNA rrna ƒ (Steffan et al., 1988; Purdy et al., 1996). Cesium chloride(cscl) density gradient centrifugation w w ƒ z w w ƒ w (Ogram et al., 1987; Porteous et al., 1991)., Steffan et al. (1988) CsCl gradient centrifucation hydroxyapatite chromatography w DNA w š, w w. ³ w CsCl gradient š sw», DNA band wì š, w» w ƒ CsCl gradient centrifugation v w. ƒ š ww p Table 2 w (Harry et al., 2000). k, PCR» w» w w v w. w DNA ƒ DNA» d.
m n m y l DNA w w š 61 ù m œ ü ew s l DNAƒ z w (Ranjard et al., 1998). 3. w Fig. 2. MCH (Magnetic Capture Hybridization)-PCR Procedure (Jacobsen., 1995). Jacobsen et al.(1995) x magnetic capture hybridization(mch) w PCR ww w œ w. Fig 2 MCH DNA sww w l p DNA w. p(biotin) t p ù DNA(single-stranded DNA) probe» w m p w ww.» (magnetic extraction) mw p DNA-DNA w z z, PCR mw s w. Chandler et al.(2000) w p w PNA(peptide nucleic acids) clamp w (oligomer) w affinity magnetic capture ww. w affinity capture p m ù ƒ m p DNA» w z. m n m y ü ky tw w y w» w w w m l š w. w w š w, DNA mw w x k tw š w. w m (soil colloids) w z DNA wwš w w (Fig. 1) Faegri et al.(1977) š Torsvik and Goksoyr(1978) w š. w w w m n m w. Hattori(1988) ƒ w Gram-positive s m œ wš, Gram-positive s œ w x. ƒ ƒ. w s DNA DNA w. w w ü DNA w s DNA w w. w w ü w DNA w w w j»ƒ j DNA w v ƒ ww. w m, n, ƒ w m l s, s w š DNA» š (Bakken et al., 1995). 3.1. m m ü e m w, w w yw w m w. x m j yw w š š, q w w w, š z š (Faegri et al., 1977; Ramsay et al., 1984; Turpin et al., 1993; Lindahl et al., 1995; Bakken et al., 1995). w w, yw z ƒ ƒƒ m m ƒ z q (Lindahl et al., 1995). yw» wì
62 Á Á y (Lindahl et al., 1995). y (Chelex 100) m z ù, Bakken(1995) m hexametaphosphate j w w (Bakken et al., 1985; Jacobsen et al., 1992). y (Chelex 100)ƒ yw w. s (lipopolysaccharides) y j (sodium cholate sodium deoxycholate), w j polyethylene glycol(peg) SDS, w PVPP yw. ù yw k (McDonald et al., 1986; Steffan et al., 1988)., SDS Chelex yw yw, s adenosine triphosphate e m w e (Bakken et al., 1995). 3.2.» s m Faegri(1977) w x. š ƒ. s Pelleting Nuclei, Chloroplast s. 6,000 rpm(6,000 g) w 2~15 w, sù w e. w w. m n m ü m 3,000 rpm 15~20 w s w. Holben et al.(1988) m ü 15 (1000 g) w fungal biomass soil debris e jš, 20 š (23,000 g)w s w. z š w 1 round w xw ƒ round m w s 10% š š w. w, w round z m s round e s tw j s w š w. w s z š w w m ƒ v w. l s ù w sw. Jacobsen(1992) y (Cation-Exchange Resin Method) m s CaCl 2 (0.1,1, or 10 mm) w s qr m (clay) ƒ s z w. w w» w (density gradient centrifugation)» š s w w» w (Bakken et al., 1985). š m 10,000-25,000 rpm(60,000 g)»» w s, w, sü». ƒ plasmic DNA» w, CsCl density gradient š w l. ù 40 š w ƒ w. š» 16» w ù šƒ CsCl w š» ù w š ( 1998). 1990 chromatography w z» CsCl š w ew, chromatography w DNA CsCl š w DNA w w DNA w š 3~4 ü ƒ j š x kit wš. ù kit w DNA 500 µg š, w m n m DNAƒ v w ƒ. DNA k m n m s w» w Percoll, metrizamide, Ficoll 400, Nycodenz, stractan ƒ gradient material w, sƒ v p t w w, š z w v w s w (Alpini et al., 1994). ù ¾ wš w s w ƒ, wš wš, s û. x ¾ s w w š, e ù x
m n m y l DNA w w š 63 Table 3. Comparison of direct and indirect extraction methods Method of extraction Direct extraction Indirect extraction DNA ƒ x w v w Application Advantage Drawback m d y tw v w DNA w w DNA z w x û w s w DNA sww w š DNA ƒ j š DNAƒ v w w û ( w w j DNA x ww ) j DNA z ƒ v w z ƒ ƒ w» w m v w. 3.3. w m DNA Table 1, yw, z mw s l w. Cesium chloride ethidium bromide equilibrium density centrifugation f j»( 48 kb) w DNA œ z w (Tiedje et al., 1988; Jacobsen et al., 1992; Courtois et al., 2003). Torsvik (1990) s w ƒ hydroxyapatite column m w w. š (urea) DNA (humic acid) w w. Steffan(1988) PVPP w (humic acid) ƒ DNA j w j, DNA z w (Steffan et al., 1988). CsCl (density centrifugation) hydroxyapatite column chromatographic purification spectrophotometer A 260/280 A 260/230 mw d DNA DNA k (Steffan et al., 1988). s w ww» agarose gel electrophoresis» w ƒ kb DNA z w. (Methanosarcina thermophila, Bacillus cereus, Mycobacterium tuberculosis, Pseudomonas aeruginosa) j DNA swwš BAC clone library w (Brosch et al., 1998; Dewar et al., 1998; Diaz-Perez et al., 1997; Rondon et al., 1999). 4. m ƒ w, w DNA w š,, w ü š (Table 3). w DNA ƒ x w v w, y wsƒ m d, š y tw v w. w w y p» z w. Leff et al.(1993) Ogram et al.(1987) w bead-beating-sds ƒ DNA ü, Tsai and Olson(1991) -w (gentle freezing-thawing lysozyme) w DNA ƒw w. w x ùš (humic acid) w. ƒ, s w DNA sww. w w w» w û DNA ü DNA ƒ ƒ DNA w š (Jacobsen and Rasmussen, 1992). w w w ù, sƒ w w š DNAƒ v w x š j DNA z ƒ v w. w ƒ j» w û.» w z x ¾ š w
64 Á Á y œw w w w. Steffan et al.(1988) z DNA w z (restriction endonuclease digestion) v w ƒ w z. CsCl z PvuII ƒ hydroxyapatite v w, EcoRI w DNA w z CsCl-hydroxyapatite m w s w DNA z w. SalI w w z qw.» w Denaturing gradient gel electrophoresis (DGGE) w (Heuer et al., 1997; Krsek et al., 1999; Maarit Niemi et al., 2001). Krsek et al.(1999) DGGE band ƒ ü. w, m w ribosomal intergenic spacer analysis(risa) mw s w w x (Martin-Laurent et al., 2001). ƒ w e w. ww kw v w š ü. w DNA w w x w xsƒ mw DNA e w w ƒ ƒ v w m. y w y» (Ecotechnopia 21 project) (051-081- 031). š x, ½ z, y, 2000, m Biomass w m sƒ, w», 2(7)135-148., w, 1998, Superscale Plasmid DNA Preparation Using Wizard Plus Maxipreps Kit, MEDLIS, 17, 136-140. Alm, E.W., Zheng, D., and Raskin, L., 2000, The presence of humic substances and DNA in RNA extracts affects hybridization results, Appl. Environ. Microbiol, 66, 4547-4554. Alpini, G., Phillips, J.O., Vroman, B., and Larusso, N.F., 1994, Recent advances in the isolation of liver cells, Hepatology, 20, 494-514. Amann, R.I., Ludwig, W., and Schleifer, K.H., 1995, Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbiol. Rev, 59, 143-169. Bakkern, L.R., 1985, Separation and purification of bacteria from soil, Appl. Environ. Microbiol. 49, 1482-1487. Bakken, L.R. and Lindahl, V., 1995, Recovery of bacterial cells from soil, Nucleic Acids in the Environment: Methods and Applications, Springer-Verlag, Heidelberg, Germany, 9-27. Brosch, R., Gordon, S.V., Billault, A., Garnier, T., Eiglmeier, K., Soravito, C., Barrell, B.G., and Cole, S.T., 1998, Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing and comparative genomics, Infect. Immun, 66, 2221-2229. Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., and Ritchie, D.A., 1992, Amplication of DNA from native populations of soil bacteria by using the polymerase chain reaction, Appl. Environ. Microbiol, 58, 3413-3416. Burgmann, H., Pesaro, M., Widmer, F., and Zeyer, J., 2001, A strategy for optimizing quality and quality of DNA extracted from soil, J. Microbiol. Methods, 45, 7-20. Chandler, D.P., Stults, J.R., Cebula, S., Schuck, B.L., Weaver, D. W., Anderson, K.K., Egholm, M., and Brockman, F.J., 2000, Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps, Appl. Environ. Microtiol, 66, 3438-3445. Courtois, S., Frostegard, A., Goransson, P., Depret, G., Jeanin, P., and Simonet, P., 2001, Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation, Environ. Microbiol, 3, 431-439. Courtois, S., Cappellano, C.M., Ball, M., Francou, F.X., Normand, P., Helynck, G., Martinez, A., Kolvek, S.J., Hopke, J., Osburne, M.S., August, P.R., Nalin, R., Guerineau, M., Jeannin, P., Simonet, P., and Pernodet, J.L., 2003, Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products, Appl. Environ. Microbiol, 69, 49-55. Curtis, T.P., Sloan, W.T., and Scannell, J.W., 2002, Estimating prokaryotic diversity and its limits, Proc. Natl. Acad. Sci. USA 99, 10494-10499. Diaz-Perez, S.V., Alatriste-Mondtagon, F., Hernandez, R., Birren, B., and Gunsalus, R.P., 1997, Bacterial artificial chromosome (BAC) library as a tool for physical mapping of the archaeon Methanosarcina thermophila TM-1, Microb. Comp. Genomics, 2, 275-286.
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