- Email: [email protected]
Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 356 (2006) 306–308 www.elsevier.com/locate/yabio
Notes & Tips
Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells Z.M. Saiyed a, C. Bochiwal a, H. Gorasia a, S.D. Telang a,¤, C.N. Ramchand b a
Department of Biochemistry, Faculty of Science, Maharaja Sayajirao University of Baroda, Sayajigunj, Vadodara 390 002, India b Kemin Nutritional Technologies (India), Chennai 600 029, India Received 22 April 2006 Available online 7 July 2006
The completion of human genome sequencing marked the end of the Wrst phase of the genomics revolution. The second phase, which has already begun, involves evaluating genetic sequences for genotyping (single-nucleotide polymorphism analysis) and allelic polymorphism analysis. This demands unprecedented capabilities for DNA puriWcation and sequencing. In addition to DNA analysis, the rapidly growing Weld of molecular diagnostics and molecular phylogeny requires a need for quick, simple, robust, and highthroughput procedures for extraction of polymerase chain reaction (PCR)1-ready DNA from diverse organisms and tissues. Traditionally, DNA extraction from mammalian cells has been a tedious and time-consuming process. As an alternative to traditional extraction protocols (e.g., phenol/ chloroform procedure), adsorption separation techniques have been applied for DNA puriWcation. The most commonly used supports to adsorb DNA include silica-based particles [1,2], glass Wbers [3], anion-exchange carrier [4–6], and modiWed magnetic beads [7–10]. During recent years, magnetic separation techniques using magnetizable solid-phase supports have become increasingly applied to a number of biological applications. Use of a magnetizable ion-exchange agarose support as a medium for the puriWcation of plasmid DNA from bacterial cell lysates has already been reported [11]. Hawkins and coworkers described the use of carboxyl-coated magnetic particles as a solid-phase support for adsorption of DNA under high-salt conditions [12]. Also, silica magnetic bead-
*
Corresponding author. E-mail address: [email protected] (S.D. Telang). 1 Abbreviations used: PCR, polymerase chain reaction; PEI, polyethylenimine; Fe3O4, magnetite; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buVered saline; PEG 6000, polyethylene glycol 6000; SDS, sodium dodecyl sulfate. 0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.06.027
based kits are commercially available (e.g., Tecan, Dynal) for rapid isolation of genomic DNA from several biological specimens. More recently, polyethylenimine (PEI)-modiWed magnetic nanobeads were employed for puriWcation of supercoiled plasmid DNA from bacterial cells [13]. Extraction of DNA using surface-modiWed magnetic beads uses only the magnetic property of the particles. However, there are reports where researchers have attempted to use magnetite (Fe3O4) for isolation of plasmid DNA and genomic DNA from bacterial cell lysates and maize kernels, respectively [14,15]. Here magnetic particles are used not only for magnetic separation but also as a solid-phase support to adsorb DNA. There are several inherent advantages envisaged for the use of such naked particles, where molecules are directly adsorbed onto the magnetic material/support. The absence of the polymer coat results in smaller particles (nano-sized, i.e., 6100 nm), thereby providing higher surface area for adsorption of biomolecules. In addition, it allows a greater response to the applied magnetic Weld; this aids in easy separation and manipulation of magnetic nanoparticles. Moreover, magnetic nanoparticles can exist as stable colloidal suspension that will not aggregate, thereby allowing uniform distribution in a reaction mixture. Therefore, in the current study, we report the applicability of magnetic nanoparticles (Fe3O4 or magnetite) as a medium for DNA puriWcation with the aim of producing a universal approach for extraction of genomic DNA from mammalian cells, namely blood leukocytes, cultured cells (HCT116), and tissues (rat liver and brain). Magnetic nanoparticles (Fe3O4) were prepared by coprecipitating divalent and trivalent iron ions by alkaline solution and treating under hydrothermal conditions [16]. After preparation, all suspensions of magnetic particles were washed repeatedly with deionized water. The yield of precipitated magnetic particles was determined by removing
Notes & Tips / Anal. Biochem. 356 (2006) 306–308
known aliquots of the suspension and drying to constant mass in an oven at 60 °C. Finally, the particles were dispersed in buVer (pH 8.0) at a suspension concentration of 10 mg/ml. The magnetic nanoparticles prepared were stable for at least 1 year at room temperature (25–30 °C) without getting agglomerated. Also, care should be taken that particles remain suspended in the buVer. Transmission electron microscopy examination revealed the mean particle size to be 40 nm. The method for isolation of genomic DNA initially was optimized using whole blood and then was investigated for its application in cultured cells (HCT116) and tissue (rat liver and brain) homogenates. The sample preparation for DNA extraction involved collection of whole blood in a tube containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Cultured cells used in this study were of colon carcinoma cell lines (HCT116), trypsinized, and adjusted to a cell density of 7 £ 106 cells/ml with phosphatebuVered saline (PBS, pH 7.4). For tissue DNA extraction, a 10% homogenate of rat liver and brain was prepared in 0.32 M buVered sucrose (pH 7.5). In a typical extraction, 30 l of sample (whole blood, cultured cell suspension, or tissue homogenate) and 30 l of 1% (w/v) sodium dodecyl sulfate (SDS) solution were added. The tube was mixed by gentle inversion two or three times and incubated at room temperature for 1 min. After incubation, 10 l of magnetic nanoparticles was added to the cell lysate, followed by the addition of 75 l of binding buVer (1.25 M sodium chloride and 10% polyethylene glycol 6000 [PEG 6000]). The suspension was mixed by inversion and allowed to stand at room temperature for 3 min. The magnetic pellet was immobilized by application of an external magnet, and the supernatant was removed. The magnetic pellet was washed with 70% ethanol and dried. Finally, the pellet was completely resuspended in 50 l of TE buVer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0), and magnetic particle-bound DNA was eluted by incubation at 65 °C with continuous agitation. We found that when using magnetic nanoparticles as solid-phase adsorbent, yields of recovered genomic DNA were up to 1.2 g per 30 l of whole blood or 2.0 £ 105 cultured cells, whereas they were approximately 1.8 and 2.0 g per 30 l of liver and brain tissue homogenate, respectively. The DNA yield in each case was estimated Xuorimetrically by Hoechst 33,258 or (preferably) by comparison of intensity of DNA bands in ethidium bromide-stained agarose gel. The molecular mass of the isolated DNA was more than 20 kb, as the band migrated at a slower rate than the 23.13-kb band of the phage/HindIII molecular mass marker (Fig. 1). In addition, the isolated DNA from all of the samples was successfully ampliWed for a 226-bp glyceraldehyde 3-phosphate dehydrogenase amplicon (Fig. 2A) with the primers as reported previously [8]. Also, the restriction digestion of isolated DNA showed no inhibitors of the enzymes present in the sample, and the DNA can be used for downstream applications (Fig. 2B). To check the robustness and reproducibility of the method, genomic
1
307
2
3
4
5
Fig. 1. Agarose gel electrophoresis of genomic DNA isolated using magnetic nanoparticles. Lane 1: DNA molecular weight marker ( phage DNA/HindIII digest); lane 2: genomic DNA isolated from rat liver homogenate; lane 3: genomic DNA isolated from rat brain homogenate; lane 4: genomic DNA isolated from cultured cells (HCT116); lane 5: genomic DNA isolated from human blood.
DNA isolation from blood and cultured cells was performed in 10 sets. The recovered genomic DNA from all 10 extractions was pooled, and spectrophotometric assessment was performed. The yield of DNA was in accordance with previous estimates, that is, approximately 12 to 15 g per 0.3 ml of blood or cultured cells. The average OD260/OD280 ratio was 1.8, indicating that the DNA was of good quality with negligible protein contamination. Also, as seen in the gel picture (Fig. 1), no low-molecular weight bands or smear were detected, indicating the absence of RNA contamination. This is in agreement with previous reports that mentioned that in presence of high-salt conditions or chaotropes, the adsorption of double-stranded DNA onto silica support and magnetite (Fe3O4) is thermodynamically favored, whereas the adsorption of proteins and singlestranded RNA is not [14,15]. The current DNA extraction method was tested for its eYciency of DNA extraction and ease of use compared with a commercially available kit (QIAamp DNA Blood Mini Kit for blood and QIAamp DNA Mini Kit for cultured cells, Qiagen). The yield of DNA extracted using magnetic nanoparticles was on average 1.3-fold greater than that using the Qiagen method. Moreover, the magnetic DNA isolation procedure can be carried out in a single microcentrifuge tube per sample, whereas the Qiagen procedure requires a number of tube transfers. The higher yield of genomic DNA obtained using magnetic nanoparticles as solid-phase support possibly may be attributed to
308
A
Notes & Tips / Anal. Biochem. 356 (2006) 306–308
1
2
3
4
5
6
7
8
9
10
Acknowledgment The Wnancial support to Z.M.S. (9/114(131)/2K2/EMRI) from the Council of ScientiWc and Industrial Research (CSIR, New Delhi, India) is gratefully acknowledged. References
B
1
2
3
4
5
6
Fig. 2. (A) Agarose gel electrophoresis of 226-bp amplicon of GAPDH gene. Lane 1: DNA molecular weight marker (100-bp ladder); lanes 2 to 4: PCR product from genomic DNA isolated from human blood using magnetic nanoparticles; lanes 5 and 6: PCR product from genomic DNA isolated from cultured cells (HCT116) using magnetic nanoparticles; lanes 7 and 8: PCR product from genomic DNA isolated from human blood using Qiagen kit; lanes 9 and 10: PCR product from genomic DNA isolated from cultured cells (HCT 116) using Qiagen kit. (B) Restriction analysis of extracted DNA. Lanes 1 and 2: undigested genomic DNA isolated using magnetic nanoparticles from human blood and cultured cells (HCT116), respectively; lanes 3 and 5: HindIII digested genomic DNA isolated from human blood using magnetic nanoparticles; lanes 4 and 6: EcoR1 digested genomic DNA isolated from cultured cells using magnetic nanoparticles.
the nano-size of the particles, which provides increased surface area for binding of DNA and creation of optimal conditions in the presence of binding buVer. In summary, the developed procedure for DNA extraction has several advantages. It is simple, quick, cheap, and robust, and it does not require the use of organic solvents. Also, the method needs only a magnet and a heating block and can be performed in any laboratory without sophisticated equipment. The procedure yields enough DNA for 30 PCR from a small quantity (30 l) of biological specimens in less than 15 min. Furthermore, the whole procedure can be accomplished in a single tube, thereby making it more amenable to automation. Currently, work is in progress for developing a universal high-throughput genomic DNA extraction system using magnetic nanoparticles as solidphase adsorbent.
[1] R. Boom, C.J. Sol, M.M. Salimans, C.L. Jansen, P.M. Wertheim-van Dillen, J. van der Noorda, Rapid and simple method for puriWcation of nucleic acids, J. Clin. Microbiol. 28 (1990) 495–503. [2] H. Tian, A.F. Huhmer, J.P. Landers, Evaluation of silica resins for direct and eYcient extraction of DNA from complex biological matrices in a miniaturized format, Anal. Biochem. 283 (2000) 175–191. [3] G.N.M. Ferreira, J.M.S. Cabral, D.M.F. Prazeres, Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatrographic supports, Biotechnol. Prog. 16 (2000) 416–424. [4] E. Thwaites, S.C. Burton, A. Lyddiatt, Impact of the physical and topographical characteristics of adsorbent solid-phases upon the Xuidised bed recovery of plasmid DNA from Escherichia coli lysates, J. Chromatogr. A 943 (2002) 77–90. [5] H.N. Endres, J.A.C. Johnson, C.A. Ross, J.K. Welp, Evaluation of an ion-exchange membrane for the puriWcation of plasmid DNA, Biotechnol. Appl. Biochem. 37 (2003) 259–266. [6] M.A. Teeters, S.E. Conrardy, B.L. Thomas, T.W. Root, E.N. Lightfoot, Adsorptive membrane chromatography for puriWcation of plasmid DNA, J. Chromatogr. A 989 (2003) 165–173. [7] K. Rudi, M. Kroken, O.J. Dahlberg, A. Deggerdal, K.S. Jakobsen, F. Larsen, Rapid universal method to isolate PCR-ready DNA using magnetic beads, BioTechniques 22 (1997) 506–511. [8] A. Deggerdal, F. Larsen, Rapid isolation of PCR-ready DNA from blood, bone marrow, and cultured cells, based on paramagnetic beads, BioTechniques 22 (1997) 554–557. [9] P.R. Levison, S.E. Badger, J. Dennis, P. Hathi, M.J. Davies, I.J. Bruce, D. Schimkat, Recent developments of magnetic beads for use in nucleic acid puriWcation, J. Chromatogr. A 816 (1998) 107–111. [10] J. Prodelalova, B. Rittich, A. Spanova, K. Petrova, M.J. Benes, Isolation of genomic DNA using magnetic cobalt ferrite and silica particles, J. Chromatogr. A 1056 (2004) 43–48. [11] M.J. Davies, D.E. Smethurst, K. Howard, M. Todd, L.M. Higgins, I.J. Bruce, Improved manufacture and application of an agarose magnetizable solid-phase support, Appl. Biochem. Biotechnol. 68 (1997) 95– 112. [12] T.L. Hawkins, T. O’Connor-Morin, A. Roy, C. Santillan, DNA puriWcation and isolation using a solid-phase, Nucleic Acids Res. 22 (1994) 4543–4544. [13] C.L. Chiang, C.S. Sung, T.F. Wu, C.Y. Chen, C.Y. Hsu, Application of superparamagnetic nanoparticles in puriWcation of plasmid DNA from bacterial cells, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 822 (2005) 54–60. [14] M.J. Davies, J.I. Taylor, N. Sachsinger, I.J. Bruce, Isolation of plasmid DNA using magnetite as a solid phase adsorbent, Anal. Biochem. 262 (1998) 92–94. [15] J.I. Taylor, C.D. Hurst, M.J. Davies, N. Sachsinger, I.J. Bruce, Application of magnetite and silica-magnetite composites to the isolation of genomic DNA, J. Chromatogr. A 890 (2000) 159–166. [16] R.V. Mehta, R.V. Upadhyay, S.W. Charles, C.N. Ramchand, Direct binding of protein to magnetic particles, Biotechnol. Tech. 11 (1997) 493–496.
Notes & Tips
Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells Z.M. Saiyed a, C. Bochiwal a, H. Gorasia a, S.D. Telang a,¤, C.N. Ramchand b a
Department of Biochemistry, Faculty of Science, Maharaja Sayajirao University of Baroda, Sayajigunj, Vadodara 390 002, India b Kemin Nutritional Technologies (India), Chennai 600 029, India Received 22 April 2006 Available online 7 July 2006
The completion of human genome sequencing marked the end of the Wrst phase of the genomics revolution. The second phase, which has already begun, involves evaluating genetic sequences for genotyping (single-nucleotide polymorphism analysis) and allelic polymorphism analysis. This demands unprecedented capabilities for DNA puriWcation and sequencing. In addition to DNA analysis, the rapidly growing Weld of molecular diagnostics and molecular phylogeny requires a need for quick, simple, robust, and highthroughput procedures for extraction of polymerase chain reaction (PCR)1-ready DNA from diverse organisms and tissues. Traditionally, DNA extraction from mammalian cells has been a tedious and time-consuming process. As an alternative to traditional extraction protocols (e.g., phenol/ chloroform procedure), adsorption separation techniques have been applied for DNA puriWcation. The most commonly used supports to adsorb DNA include silica-based particles [1,2], glass Wbers [3], anion-exchange carrier [4–6], and modiWed magnetic beads [7–10]. During recent years, magnetic separation techniques using magnetizable solid-phase supports have become increasingly applied to a number of biological applications. Use of a magnetizable ion-exchange agarose support as a medium for the puriWcation of plasmid DNA from bacterial cell lysates has already been reported [11]. Hawkins and coworkers described the use of carboxyl-coated magnetic particles as a solid-phase support for adsorption of DNA under high-salt conditions [12]. Also, silica magnetic bead-
*
Corresponding author. E-mail address: [email protected] (S.D. Telang). 1 Abbreviations used: PCR, polymerase chain reaction; PEI, polyethylenimine; Fe3O4, magnetite; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buVered saline; PEG 6000, polyethylene glycol 6000; SDS, sodium dodecyl sulfate. 0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.06.027
based kits are commercially available (e.g., Tecan, Dynal) for rapid isolation of genomic DNA from several biological specimens. More recently, polyethylenimine (PEI)-modiWed magnetic nanobeads were employed for puriWcation of supercoiled plasmid DNA from bacterial cells [13]. Extraction of DNA using surface-modiWed magnetic beads uses only the magnetic property of the particles. However, there are reports where researchers have attempted to use magnetite (Fe3O4) for isolation of plasmid DNA and genomic DNA from bacterial cell lysates and maize kernels, respectively [14,15]. Here magnetic particles are used not only for magnetic separation but also as a solid-phase support to adsorb DNA. There are several inherent advantages envisaged for the use of such naked particles, where molecules are directly adsorbed onto the magnetic material/support. The absence of the polymer coat results in smaller particles (nano-sized, i.e., 6100 nm), thereby providing higher surface area for adsorption of biomolecules. In addition, it allows a greater response to the applied magnetic Weld; this aids in easy separation and manipulation of magnetic nanoparticles. Moreover, magnetic nanoparticles can exist as stable colloidal suspension that will not aggregate, thereby allowing uniform distribution in a reaction mixture. Therefore, in the current study, we report the applicability of magnetic nanoparticles (Fe3O4 or magnetite) as a medium for DNA puriWcation with the aim of producing a universal approach for extraction of genomic DNA from mammalian cells, namely blood leukocytes, cultured cells (HCT116), and tissues (rat liver and brain). Magnetic nanoparticles (Fe3O4) were prepared by coprecipitating divalent and trivalent iron ions by alkaline solution and treating under hydrothermal conditions [16]. After preparation, all suspensions of magnetic particles were washed repeatedly with deionized water. The yield of precipitated magnetic particles was determined by removing
Notes & Tips / Anal. Biochem. 356 (2006) 306–308
known aliquots of the suspension and drying to constant mass in an oven at 60 °C. Finally, the particles were dispersed in buVer (pH 8.0) at a suspension concentration of 10 mg/ml. The magnetic nanoparticles prepared were stable for at least 1 year at room temperature (25–30 °C) without getting agglomerated. Also, care should be taken that particles remain suspended in the buVer. Transmission electron microscopy examination revealed the mean particle size to be 40 nm. The method for isolation of genomic DNA initially was optimized using whole blood and then was investigated for its application in cultured cells (HCT116) and tissue (rat liver and brain) homogenates. The sample preparation for DNA extraction involved collection of whole blood in a tube containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Cultured cells used in this study were of colon carcinoma cell lines (HCT116), trypsinized, and adjusted to a cell density of 7 £ 106 cells/ml with phosphatebuVered saline (PBS, pH 7.4). For tissue DNA extraction, a 10% homogenate of rat liver and brain was prepared in 0.32 M buVered sucrose (pH 7.5). In a typical extraction, 30 l of sample (whole blood, cultured cell suspension, or tissue homogenate) and 30 l of 1% (w/v) sodium dodecyl sulfate (SDS) solution were added. The tube was mixed by gentle inversion two or three times and incubated at room temperature for 1 min. After incubation, 10 l of magnetic nanoparticles was added to the cell lysate, followed by the addition of 75 l of binding buVer (1.25 M sodium chloride and 10% polyethylene glycol 6000 [PEG 6000]). The suspension was mixed by inversion and allowed to stand at room temperature for 3 min. The magnetic pellet was immobilized by application of an external magnet, and the supernatant was removed. The magnetic pellet was washed with 70% ethanol and dried. Finally, the pellet was completely resuspended in 50 l of TE buVer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0), and magnetic particle-bound DNA was eluted by incubation at 65 °C with continuous agitation. We found that when using magnetic nanoparticles as solid-phase adsorbent, yields of recovered genomic DNA were up to 1.2 g per 30 l of whole blood or 2.0 £ 105 cultured cells, whereas they were approximately 1.8 and 2.0 g per 30 l of liver and brain tissue homogenate, respectively. The DNA yield in each case was estimated Xuorimetrically by Hoechst 33,258 or (preferably) by comparison of intensity of DNA bands in ethidium bromide-stained agarose gel. The molecular mass of the isolated DNA was more than 20 kb, as the band migrated at a slower rate than the 23.13-kb band of the phage/HindIII molecular mass marker (Fig. 1). In addition, the isolated DNA from all of the samples was successfully ampliWed for a 226-bp glyceraldehyde 3-phosphate dehydrogenase amplicon (Fig. 2A) with the primers as reported previously [8]. Also, the restriction digestion of isolated DNA showed no inhibitors of the enzymes present in the sample, and the DNA can be used for downstream applications (Fig. 2B). To check the robustness and reproducibility of the method, genomic
1
307
2
3
4
5
Fig. 1. Agarose gel electrophoresis of genomic DNA isolated using magnetic nanoparticles. Lane 1: DNA molecular weight marker ( phage DNA/HindIII digest); lane 2: genomic DNA isolated from rat liver homogenate; lane 3: genomic DNA isolated from rat brain homogenate; lane 4: genomic DNA isolated from cultured cells (HCT116); lane 5: genomic DNA isolated from human blood.
DNA isolation from blood and cultured cells was performed in 10 sets. The recovered genomic DNA from all 10 extractions was pooled, and spectrophotometric assessment was performed. The yield of DNA was in accordance with previous estimates, that is, approximately 12 to 15 g per 0.3 ml of blood or cultured cells. The average OD260/OD280 ratio was 1.8, indicating that the DNA was of good quality with negligible protein contamination. Also, as seen in the gel picture (Fig. 1), no low-molecular weight bands or smear were detected, indicating the absence of RNA contamination. This is in agreement with previous reports that mentioned that in presence of high-salt conditions or chaotropes, the adsorption of double-stranded DNA onto silica support and magnetite (Fe3O4) is thermodynamically favored, whereas the adsorption of proteins and singlestranded RNA is not [14,15]. The current DNA extraction method was tested for its eYciency of DNA extraction and ease of use compared with a commercially available kit (QIAamp DNA Blood Mini Kit for blood and QIAamp DNA Mini Kit for cultured cells, Qiagen). The yield of DNA extracted using magnetic nanoparticles was on average 1.3-fold greater than that using the Qiagen method. Moreover, the magnetic DNA isolation procedure can be carried out in a single microcentrifuge tube per sample, whereas the Qiagen procedure requires a number of tube transfers. The higher yield of genomic DNA obtained using magnetic nanoparticles as solid-phase support possibly may be attributed to
308
A
Notes & Tips / Anal. Biochem. 356 (2006) 306–308
1
2
3
4
5
6
7
8
9
10
Acknowledgment The Wnancial support to Z.M.S. (9/114(131)/2K2/EMRI) from the Council of ScientiWc and Industrial Research (CSIR, New Delhi, India) is gratefully acknowledged. References
B
1
2
3
4
5
6
Fig. 2. (A) Agarose gel electrophoresis of 226-bp amplicon of GAPDH gene. Lane 1: DNA molecular weight marker (100-bp ladder); lanes 2 to 4: PCR product from genomic DNA isolated from human blood using magnetic nanoparticles; lanes 5 and 6: PCR product from genomic DNA isolated from cultured cells (HCT116) using magnetic nanoparticles; lanes 7 and 8: PCR product from genomic DNA isolated from human blood using Qiagen kit; lanes 9 and 10: PCR product from genomic DNA isolated from cultured cells (HCT 116) using Qiagen kit. (B) Restriction analysis of extracted DNA. Lanes 1 and 2: undigested genomic DNA isolated using magnetic nanoparticles from human blood and cultured cells (HCT116), respectively; lanes 3 and 5: HindIII digested genomic DNA isolated from human blood using magnetic nanoparticles; lanes 4 and 6: EcoR1 digested genomic DNA isolated from cultured cells using magnetic nanoparticles.
the nano-size of the particles, which provides increased surface area for binding of DNA and creation of optimal conditions in the presence of binding buVer. In summary, the developed procedure for DNA extraction has several advantages. It is simple, quick, cheap, and robust, and it does not require the use of organic solvents. Also, the method needs only a magnet and a heating block and can be performed in any laboratory without sophisticated equipment. The procedure yields enough DNA for 30 PCR from a small quantity (30 l) of biological specimens in less than 15 min. Furthermore, the whole procedure can be accomplished in a single tube, thereby making it more amenable to automation. Currently, work is in progress for developing a universal high-throughput genomic DNA extraction system using magnetic nanoparticles as solidphase adsorbent.
[1] R. Boom, C.J. Sol, M.M. Salimans, C.L. Jansen, P.M. Wertheim-van Dillen, J. van der Noorda, Rapid and simple method for puriWcation of nucleic acids, J. Clin. Microbiol. 28 (1990) 495–503. [2] H. Tian, A.F. Huhmer, J.P. Landers, Evaluation of silica resins for direct and eYcient extraction of DNA from complex biological matrices in a miniaturized format, Anal. Biochem. 283 (2000) 175–191. [3] G.N.M. Ferreira, J.M.S. Cabral, D.M.F. Prazeres, Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatrographic supports, Biotechnol. Prog. 16 (2000) 416–424. [4] E. Thwaites, S.C. Burton, A. Lyddiatt, Impact of the physical and topographical characteristics of adsorbent solid-phases upon the Xuidised bed recovery of plasmid DNA from Escherichia coli lysates, J. Chromatogr. A 943 (2002) 77–90. [5] H.N. Endres, J.A.C. Johnson, C.A. Ross, J.K. Welp, Evaluation of an ion-exchange membrane for the puriWcation of plasmid DNA, Biotechnol. Appl. Biochem. 37 (2003) 259–266. [6] M.A. Teeters, S.E. Conrardy, B.L. Thomas, T.W. Root, E.N. Lightfoot, Adsorptive membrane chromatography for puriWcation of plasmid DNA, J. Chromatogr. A 989 (2003) 165–173. [7] K. Rudi, M. Kroken, O.J. Dahlberg, A. Deggerdal, K.S. Jakobsen, F. Larsen, Rapid universal method to isolate PCR-ready DNA using magnetic beads, BioTechniques 22 (1997) 506–511. [8] A. Deggerdal, F. Larsen, Rapid isolation of PCR-ready DNA from blood, bone marrow, and cultured cells, based on paramagnetic beads, BioTechniques 22 (1997) 554–557. [9] P.R. Levison, S.E. Badger, J. Dennis, P. Hathi, M.J. Davies, I.J. Bruce, D. Schimkat, Recent developments of magnetic beads for use in nucleic acid puriWcation, J. Chromatogr. A 816 (1998) 107–111. [10] J. Prodelalova, B. Rittich, A. Spanova, K. Petrova, M.J. Benes, Isolation of genomic DNA using magnetic cobalt ferrite and silica particles, J. Chromatogr. A 1056 (2004) 43–48. [11] M.J. Davies, D.E. Smethurst, K. Howard, M. Todd, L.M. Higgins, I.J. Bruce, Improved manufacture and application of an agarose magnetizable solid-phase support, Appl. Biochem. Biotechnol. 68 (1997) 95– 112. [12] T.L. Hawkins, T. O’Connor-Morin, A. Roy, C. Santillan, DNA puriWcation and isolation using a solid-phase, Nucleic Acids Res. 22 (1994) 4543–4544. [13] C.L. Chiang, C.S. Sung, T.F. Wu, C.Y. Chen, C.Y. Hsu, Application of superparamagnetic nanoparticles in puriWcation of plasmid DNA from bacterial cells, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 822 (2005) 54–60. [14] M.J. Davies, J.I. Taylor, N. Sachsinger, I.J. Bruce, Isolation of plasmid DNA using magnetite as a solid phase adsorbent, Anal. Biochem. 262 (1998) 92–94. [15] J.I. Taylor, C.D. Hurst, M.J. Davies, N. Sachsinger, I.J. Bruce, Application of magnetite and silica-magnetite composites to the isolation of genomic DNA, J. Chromatogr. A 890 (2000) 159–166. [16] R.V. Mehta, R.V. Upadhyay, S.W. Charles, C.N. Ramchand, Direct binding of protein to magnetic particles, Biotechnol. Tech. 11 (1997) 493–496.