- Email: [email protected]
Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification
JOURNALOF BIOSCIENCE ANDBIOENGINEERING Vol. 91, No. 5, 500-503. 2001
Development of a Novel Method for Operating Magnetic Particles, Magtration Technology, and Its Use for Automating Nucleic Acid Purification KIMIMICHI OBATA,’ OSAMU SEGAWA,’ MITSURU YAKABE,’ YOSHIKAZU ISHIDA,2 TOSHIHIRO KUROITA,2 KATSUNORI IKEDA,2 BUNSEI KAWAKAMI,2 YOSHIHISA KAWAMURA,2 MASAFUMI YOHDA,3 TADASHI MATSUNAGA,3 AND HIDEJI TAJIMA’* Precision System Science Co. Ltd., 88 Kamihongo, Matsudo, Chiba 271-0064,’ Tsuruga Institute of Biotechnology, Toyobo Co. Ltd., IO-24 Toyo-cho, Tsuruga, Fukui 914-0047,2 and Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588,’ Japan Received 4 September 2OOO/Accepted 14 February 2001
Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and puritication using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods. [Key words: magnetic separation,
beads, nucleic acid, automation]
Recent advances in genome analysis have made genetic diagnosis an important tool in clinical medicine. Currently, a need exists to automate the various steps in genetic diagnosis, such as nucleic acid purification, sequencing, PCR and detection of DNA. Automation minimizes manual labor and provides more reproducible results, possibly also of higher quality. Automation is also important for accelerating large-scale sequencing projects. Many instruments have been developed to automate PCR amplification, sequencing reactions and detection of nucleic acids (l-4). However, full automation has not been achieved probably because of the difficulty in automating DNA extraction and purification processes. It is especially difficult to extract and purify nucleic acids from samples to detect infectious bacteria or viruses because contamination of instruments or cross-contamination among samples occurs easily. Conventional DNA extraction and purification usually requires phenol extraction and ethanol precipitation, which are performed by centrifugation, but it is difficult to automate genetic manipulation processes using centrifugation. Although some automated instruments for nucleic acid purification by centrifugation have been commercialized, they are used only for limited purposes. The idea of using magnetic separation techniques in molecular biology has developed primarily as the result of the successful removal by magnetic separation of tumor cells from bone marrow and the isolation of lymphoid cells from peripheral blood (5). Hultman and coworkers have developed a solid phase sequence which enables single-stranded DNA to be purified from PCR products by magnetic separation (6, 7). In their method, the target DNA is amplified with biotinylated primers and captured by streptavidin-linked magnetic beads. The
bound double-stranded DNA is denatured by alkaline treatment and single-stranded DNA is obtained. Magnetic beads have since been used for extraction and purification of nucleic acids. In combination with an efficient automation system to operate magnetic particles, the use of magnetic beads is expected to enable concentration and purification of nucleic acids in a fast and convenient manner. Without such a system, magnetic separation is inconvenient compared with conventional methods using centrifugation. Thus, the use of magnetic beads in molecular biology has been limited. In a conventional magnetic separator, permanent magnets are placed under the microtiter plate or tubes, and are moved up and down using a mechanical system (8, 9). To capture the magnetic beads, the magnets move into the immediate vicinity of all wells or tubes and rapidly separate the beads to the sides of the wells. Magnetic separation is terminated by moving the magnets away from the wells. Magnetic separation can be performed using a commercially available robotic workstation equipped with a magnetic separator as described above. However, it is difficult to achieve a high recovery of magnetic particles using this system because recovery of the liquid phase from the well is not easy. It is also difficult to prevent the scattering and contamination of samples during resuspension of samples into the wells. We have developed a novel method for operating magnetic particles, termed Magtration Technology. Separation and resuspension of magnetic particles is performed within a disposable tip. Thus, the risk of cross contamination among samples is reduced and the yield is comparable to those of conventional methods. In addition, this method lends itself to easy automation and upgrading. Automated instruments based on Magtration Technology were developed and the efficiency of automated DNA/RNA/plasmid purification by Magtration Tech-
* Corresponding author. e-mail: [email protected] phone: +81-(0)47-303-4800 fax: +81-(0)47-303-4810 500
AUTOMATED NUCLEIC ACID PURIFICATION
VOL. 91, 2001
nology was compared
to that of manual
MATERIALS
preparation.
AND METHODS
Strain, plasmid and reagents Escherichia coli JM 109 was used for propagating plasmid pUC18. HeLa cells and HL60 cells were cultured in D-MEM containing 10% FCS and 50 pg/ml of gentamycin. Restriction enzymes, rTaq polymerase, M-MLV RTase (RNase HP), and RNase inhibitor were products of Toyobo. Purification of genomic DNA, total RNA and plasmids Purification of genomic DNA, total RNA and plasmids using the automated system (MFX-2000) was performed using kits for nucleic acid purification by Magtration Technology (MagExtractor-Genome, MagExtractor-RNA and MagExtractor-plasmid (Toyobo, Tsuruga, Fukui)). MagExtractor-Genome and MagExtractor-RNA were developed based on the Boom method (lo), while MagExtractor-Plasmid is based on the alkaline SDS method (11) and purification of plasmids using glass beads (12). One hundred ~1 of whole blood was used for purification of genomic DNA. E. coli harboring pUC18 was cultured up to OD 9, and 1.2 ml was used for plasmid extraction. Manual preparation of total RNA from HeLa cells was carried out according to the AGPC method (13). The oligonucleotide primers, 5’PCR amplification TGT-TCA-C’IT-GTG-CCC-TGA-CT-3’ and S-AGC-AATCAG-TGA-GGA-ATC-AG-3’, were used for amplifying exon 5 (310 bp) of the human ~53 gene. Exon 8 (445 bp) was amplified using the primers 5’-TTG-GGA-GTAGAT-GGA-GCC-T-3’ and 5’-AGT-GTT-AGA-CTG-GAAACT-TT-3’. One-twentieth of the DNA extracted from 100 ~1 of whole blood was used for PCR amplification in 50 ~1 of reaction solution containing dNTPs (0.2 ,YM each), MgClz (1.5 mM), primers (0.2 PM each), and 1.25 U of rTaq DNA polymerase. After 30 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min in a thermal cycler (PJ2000, Perkin Elmer, Foster City, California, USA), the reaction solution was analyzed by agarose gel electrophoresis. RT-PCR A fragment of the transferrin receptor mRNA (2115 bases) was amplified by RT PCR using an F-primer (5’-CCA-CCA-TCT-CGG-TCA-TCA-GGA-TTGCCT3’) and an R-primer (5’~AAA-GTC-TGA-CAAACT-GAG-TCT-GCA-AC-3’). One /lg of total RNA, 1 mM each of dATP, dGTP, dCTP and dTTP, 20U of M-MLV RTase (RNase H-), 1OU of RNase inhibitor and the R primer (1.25 PM) were mixed in a final volume of 20 ~1. Reverse transcription was performed at 42°C for 30 min, and then the reaction mixture was heated at 99°C for 5 min. The reaction product was subjected to PCR in a lOO-~1 reaction solution containing 2.5 U of rTaq DNA polymerase, and 0.125 PM of each primer. After 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 2min, the reaction solution was analyzed by agarose gel electrophoresis. RESULTS
BY MAGTRATION TECHNOLOGY
501
section, magnetic separation is achieved during the sucking or dispensing of a suspension of magnetic particles by placing a magnet against the outside of the tip. Resuspension of the trapped magnetic particles is easily performed by sucking and dispensing the resuspension buffer with the magnet moved away from the tip. The inner diameter of the disposable tip for an intermediate region with a capacity of l-ml is only 2mm. Thus, the magnetic field in the tip is sufficiently strong to trap magnetic particles. The magnetic particle suspension passes through the high magnetic field region for a length of 15 mm, and almost all the magnetic particles are captured. Each sample is handled in the disposable tip. In the nucleic acid purification process, the buffer or reagent required is dispensed into the tubes first to reduce the possibility of cross-contamination between samples. Moreover, the separation and resuspension of magnetic particles are performed within the disposable tip. Thus, the risk of cross-contamination among the samples is greatly reduced and recovery is high compared with the conventional method. In addition, this method is amenable to automation and upgrading. Using Magtration Technology, two distinct instruments, SX-OG (Precision System Science, Matsudo, Chiba) and MFX-2000 (Toyobo), for the automation of nucleic acid extraction and purification were developed. The main components of the instruments are almost identical. SX-OG is controlled by a personal computer, and can be used for various purposes by changing protocols and parameters. MFX-2000 is designed especially for nucleic acid purification. Protocols for nucleic acid purification are preinstalled and the settings are optimized. Both instruments handle 24 samples successively. The instruments are composed of only two units, a stage on which to place the disposable tubes, tips and reagents, and the Magtration unit, a liquid dispensing unit with a magnet (Fig. 1). Thus, the system is very simple and inexpensive compared with other systems involving magnetic separation. Total DNA, RNA and plasNucleic acid extraction mids were extracted and purified by reagents developed for Magtration Technology based on the Boom method (Total DNA and RNA) (10) or by a glass-bead-based plasmid purification method (12). The basic procedure is outlined in Fig. 2. Cells or bacteria were lysed by
AND DISCUSSION
In MagtraMagtration Technology and instruments tion Technology, magnetic separation is performed in a specially designed disposable tip. The tip has three parts; a reservoir to aspirate the reaction mixture, a thin tip end, and an intermediate section. In the intermediate
FIG. 1. nology.
Tip and magnetic separation unit for magtration tech-
502
J. BIOSCI.BIOENG..
OBATA ET AL.
Sample FIG. 2.
Cell Lysis
Addition of magnetic particles
Release of nucleic acids
Recovery of nucleic acids
Schematic procedure for nucleic acid purification by magtration technology.
chaotropic reagents, causing the released nucleic acids which then absorbed on magnetic silica beads under high-ionic-strength conditions. By changing the ionic strength, each nucleic acid could be selectively captured. Unbound proteins, nucleic acids and other small cellular molecules were removed by washing with 70% ethanol. Bound nucleic acid could be eluted from the beads with Hz0 or low ionic strength buffer. This series of processes was complete within 10 min per sample. Extraction of total DNA from human whole blood The yield of genomic DNA from and HeLa cells human whole blood was 2 ~g/lOO ~1. The A26O/A280 ratio of the purified DNA was 1.8kO.l. The yield and purity were comparable to values for other commercially available DNA extraction kits (Fig. 3A). The extracted genomic DNA was subjected to PCR amplification. As shown in Fig. 3B, exon 5 and exon 8 of the human p53 gene were successfully amplified from the purified genomic DNA. The yield of genomic DNA from HeLa cells was 2.9
(A) 12
Magnetic capture of nucleic acids
(B) 123
pg/5 x lo5 cells and the A260/A280 ratio of the purified DNA was 1.84. Although the yield measured from the OD was significantly low compared with those obtained using other methods (8.1 to 9.7 ,ug), the yield estimated from the results of gel electrophoresis was not (data not shown). This result suggests that the purity of the DNA purified by this method is relatively high compared with that of DNA obtained using other methods. Purification of total RNA and amplification of cDNA encoding the transfer& receptor by RT-PCR Total RNA was purified from HeLa cells and HL60 cells. The yield and purity of the RNA purified from 2 x IO6 HL60 cells were 18 lug and 1.87 (A26O/A280), respectively. The values were comparable to those of manual extraction using the AGPC method (13 pg and 1.9 (A260IA280)). Figure 4A shows the results of agarose gel electrophoresis of the purified RNA. The 18s and 28s rRNAs were clearly observed. Total RNA extracted from 2 x 106 HeLa cells was used for RT-PCR targeting the transferrin receptor mRNA. The transferrin receptor gene was amplified only when reverse transcription was performed prior to PCR (Fig. 4B) Plasmid purification and restriction digestion E.
(4
(W 1
2
1
2
3
.Exon 8 (445 bp) 5 (310 bp)
FIG. 3. Purification of total genomic DNA and PCR amplification. (A) 1, Molecular weight markers 1 phage DNA digested by PstI; 2, total genomic DNA from a whole blood cell purified by magtration technology. (B) 1, Molecular weight markers 1 phage DNA digested by PstI; 2, PCR-amplified exon 5 of the ~53 gene from total genomic DNA of a whole blood cell purified by magtration technology; 3, PCR-amplified exon 8 of the p53 gene from total genomic DNA of a whole blood cell purified by magtration technology.
FIG. 4. Purification and RT-PCR of total RNA of the human transferrin receptor. (A) 1, Total RNA from HL60 cells purified manually by the AGPC method; 2, total RNA from HL 60 cells purified by magtration technology. (B) 1, Molecular weight markers, R phage DNA digested by ZfindIII; 2, RT-PCR product obtained with RTase; 3, RT-PCR product obtained without RTase.
VOL. 91, 2001
AUTOMATED NUCLEIC ACID PURIFICATION
BY MAGTRATION TECHNOLOGY
503
no less than those for manually purified nucleic acids. The main shortcoming of the system is that it is timeconsuming to purify many samples. Since the instrument is equipped with only one nozzle, purification is individually performed. Thus, it takes about 4 h to purify nucleic acid from 24 samples. This problem could be easily overcome by developing an instrument equipped with multiple nozzles. We have already developed systems with 6, 12 and 96 nozzles. The 6-nozzle instrument reduced the time required for purification 6-fold, with 6 samples being treated in 10min. Nucleotide purification from 48 samples could be performed in only 80 min. REFERENCES
FIG. 5. Purification of plasmid by magtration technology and its restriction digestion. 1, Molecular weight markers, A phage DNA digested by iYindII1; 2, plasmids, pUCl8 purified by magtration technology; 3, KpnI-digested pUCl8 purified by magtration technology; 4, &a-I-digested pUCl8 purified by magtration technology.
JM109 harboring plasmid pUC18 was cultured in 1.2 ml of L-broth and used for plasmid purification by MFX2000. E. coli cells were lysed by alkaline/SDS. Following neutralization, the cell debris of genomic DNA, denatured protein and other polymers were captured with magnetic beads and removed by magnetic separation. Silica magnetic beads were then added to the clear lysate, and the plasmid was purified. The yield of plasmid from E. coli was 3 to 6 pg/1.2 ml of broth (JM109/pUC18) and the A260/A280 ratio was 1.8fO.l. There was almost no contamination by genomic DNA or RNA. The purified plasmid was completely digested by KpnI and Z&z1 (Fig. 5). The yield and purity of the extracted plasmid were no less than those of manually purified plasmid. The purified plasmid could be used directly for sequencing as a template. To confirm that no cross-contamination among samples occurs during nucleic acid purification by Magtration Technology, 100 ~1 of human serum containing the HCV virus at a concentration of 107/ml and the same volume of human serum without the virus were alternatively placed in 24 sample stands of the MFX2000, and nucleic acid purification was performed. The purified nucleic acid was eluted in Hz0 and subjected to HCV RNA detection by RT PCR using the Amplicore-HCV kit (Nippon Roche, Tokyo). All 12 nucleic acids purified from negative sera were judged to be HCV-RNA-negative while other nucleic acids obtained from positive sera gave positive signals (unpublished data). The present results clearly demonstrate that Magtration Technology enables automated purification of nucleic acids from various sources. The automation instrument performed with high efficiency in the purification of total genomic DNA, total RNA and plasmids. The yield and purity of the purified nucleic acids were coli
1. Earley, J. J., Kuivaniemi, H., Prockop, D. J., and Tromp, G.: Robotic automation of dideoxyribonucleotide sequencing reactions. Biotechniques, 17, 156-165 (1994). 2. Caiflat-Zucman, S., Car&on, H. J., Costantino, F., Cot, S., and Bach, J.F.: Automation of large-scale HLA oligotyping using a robotic workstation. Biotechniques, 15, 526-531 (1993). 3. Fowler, A. and Sutton, L. D.: Robot-controlled operation of a PCR system thermal cycler. Am. Biotechnol. Lab., 12, 17 (1994). 4. Fujita, hi., Usnl, S., Klyama, M., Kambara, H., Murakawa, K., Soauki, S., Sambe, H., and Takachi, K.: Chemical robot for enzymatic reactions and extraction processes of DNA in DNA sequence analysis. Biotechniques, 9, 584-591 (1990). 5. Lea, T., Vartdal, F., Nustad, K., Funderud, S., Berge, A., Ellingsen, T., S&mid, R., Stenstad, P., and Ugelstad, J.: Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro. J. Mol. Recognit., 1, 9-18 (1988). 6. Hultman, T., Stahl, S., Hornes, E., and Uhlen, M.: Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucl. Acids Res., 18, 51075112 (1989). 7. Hultman, T., Bergh, S., Moks, T., and Uhlen, M.: Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. Biotechniques, 10, 84-93 (1991). nonradioactive 8. Rolfs, A. and Weber, I.: Fully-automated, solid-phase sequencing of genomic DNA obtained from PCR. Biotechniques, 17, 782-787 (1994). 9. Fry, G., Lachenmeier, E., Mayrand, E., Giusti, B., Fisher, J., Johnston-Dow, L., Catheart, R., Finne, E., and Kifaas, L.: A new approach to template purification for sequencing applications using paramagnetic particles. Biotechniques, 13, 124-131 (1992). 10. Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P.M., and van der Noordaa, J.: Rapid and simple method for purification of nucleic acids. J. Clin. Microbial., 28, 495-503 (1990). 11. Biinboim, H. C. and Doly, J.: A rapid alkaline extraction for screening recombinant plasmid DNA. Nucl. Acids Res., 7, 1513-1523 (1979) 12. Marko, M. A., Chippeffield, R., and Birnboim, H. C.: A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Ana. Biochem., 121,382-387 (1982) 13. Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156-159 (1987).
Development of a Novel Method for Operating Magnetic Particles, Magtration Technology, and Its Use for Automating Nucleic Acid Purification KIMIMICHI OBATA,’ OSAMU SEGAWA,’ MITSURU YAKABE,’ YOSHIKAZU ISHIDA,2 TOSHIHIRO KUROITA,2 KATSUNORI IKEDA,2 BUNSEI KAWAKAMI,2 YOSHIHISA KAWAMURA,2 MASAFUMI YOHDA,3 TADASHI MATSUNAGA,3 AND HIDEJI TAJIMA’* Precision System Science Co. Ltd., 88 Kamihongo, Matsudo, Chiba 271-0064,’ Tsuruga Institute of Biotechnology, Toyobo Co. Ltd., IO-24 Toyo-cho, Tsuruga, Fukui 914-0047,2 and Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588,’ Japan Received 4 September 2OOO/Accepted 14 February 2001
Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and puritication using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods. [Key words: magnetic separation,
beads, nucleic acid, automation]
Recent advances in genome analysis have made genetic diagnosis an important tool in clinical medicine. Currently, a need exists to automate the various steps in genetic diagnosis, such as nucleic acid purification, sequencing, PCR and detection of DNA. Automation minimizes manual labor and provides more reproducible results, possibly also of higher quality. Automation is also important for accelerating large-scale sequencing projects. Many instruments have been developed to automate PCR amplification, sequencing reactions and detection of nucleic acids (l-4). However, full automation has not been achieved probably because of the difficulty in automating DNA extraction and purification processes. It is especially difficult to extract and purify nucleic acids from samples to detect infectious bacteria or viruses because contamination of instruments or cross-contamination among samples occurs easily. Conventional DNA extraction and purification usually requires phenol extraction and ethanol precipitation, which are performed by centrifugation, but it is difficult to automate genetic manipulation processes using centrifugation. Although some automated instruments for nucleic acid purification by centrifugation have been commercialized, they are used only for limited purposes. The idea of using magnetic separation techniques in molecular biology has developed primarily as the result of the successful removal by magnetic separation of tumor cells from bone marrow and the isolation of lymphoid cells from peripheral blood (5). Hultman and coworkers have developed a solid phase sequence which enables single-stranded DNA to be purified from PCR products by magnetic separation (6, 7). In their method, the target DNA is amplified with biotinylated primers and captured by streptavidin-linked magnetic beads. The
bound double-stranded DNA is denatured by alkaline treatment and single-stranded DNA is obtained. Magnetic beads have since been used for extraction and purification of nucleic acids. In combination with an efficient automation system to operate magnetic particles, the use of magnetic beads is expected to enable concentration and purification of nucleic acids in a fast and convenient manner. Without such a system, magnetic separation is inconvenient compared with conventional methods using centrifugation. Thus, the use of magnetic beads in molecular biology has been limited. In a conventional magnetic separator, permanent magnets are placed under the microtiter plate or tubes, and are moved up and down using a mechanical system (8, 9). To capture the magnetic beads, the magnets move into the immediate vicinity of all wells or tubes and rapidly separate the beads to the sides of the wells. Magnetic separation is terminated by moving the magnets away from the wells. Magnetic separation can be performed using a commercially available robotic workstation equipped with a magnetic separator as described above. However, it is difficult to achieve a high recovery of magnetic particles using this system because recovery of the liquid phase from the well is not easy. It is also difficult to prevent the scattering and contamination of samples during resuspension of samples into the wells. We have developed a novel method for operating magnetic particles, termed Magtration Technology. Separation and resuspension of magnetic particles is performed within a disposable tip. Thus, the risk of cross contamination among samples is reduced and the yield is comparable to those of conventional methods. In addition, this method lends itself to easy automation and upgrading. Automated instruments based on Magtration Technology were developed and the efficiency of automated DNA/RNA/plasmid purification by Magtration Tech-
* Corresponding author. e-mail: [email protected] phone: +81-(0)47-303-4800 fax: +81-(0)47-303-4810 500
AUTOMATED NUCLEIC ACID PURIFICATION
VOL. 91, 2001
nology was compared
to that of manual
MATERIALS
preparation.
AND METHODS
Strain, plasmid and reagents Escherichia coli JM 109 was used for propagating plasmid pUC18. HeLa cells and HL60 cells were cultured in D-MEM containing 10% FCS and 50 pg/ml of gentamycin. Restriction enzymes, rTaq polymerase, M-MLV RTase (RNase HP), and RNase inhibitor were products of Toyobo. Purification of genomic DNA, total RNA and plasmids Purification of genomic DNA, total RNA and plasmids using the automated system (MFX-2000) was performed using kits for nucleic acid purification by Magtration Technology (MagExtractor-Genome, MagExtractor-RNA and MagExtractor-plasmid (Toyobo, Tsuruga, Fukui)). MagExtractor-Genome and MagExtractor-RNA were developed based on the Boom method (lo), while MagExtractor-Plasmid is based on the alkaline SDS method (11) and purification of plasmids using glass beads (12). One hundred ~1 of whole blood was used for purification of genomic DNA. E. coli harboring pUC18 was cultured up to OD 9, and 1.2 ml was used for plasmid extraction. Manual preparation of total RNA from HeLa cells was carried out according to the AGPC method (13). The oligonucleotide primers, 5’PCR amplification TGT-TCA-C’IT-GTG-CCC-TGA-CT-3’ and S-AGC-AATCAG-TGA-GGA-ATC-AG-3’, were used for amplifying exon 5 (310 bp) of the human ~53 gene. Exon 8 (445 bp) was amplified using the primers 5’-TTG-GGA-GTAGAT-GGA-GCC-T-3’ and 5’-AGT-GTT-AGA-CTG-GAAACT-TT-3’. One-twentieth of the DNA extracted from 100 ~1 of whole blood was used for PCR amplification in 50 ~1 of reaction solution containing dNTPs (0.2 ,YM each), MgClz (1.5 mM), primers (0.2 PM each), and 1.25 U of rTaq DNA polymerase. After 30 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min in a thermal cycler (PJ2000, Perkin Elmer, Foster City, California, USA), the reaction solution was analyzed by agarose gel electrophoresis. RT-PCR A fragment of the transferrin receptor mRNA (2115 bases) was amplified by RT PCR using an F-primer (5’-CCA-CCA-TCT-CGG-TCA-TCA-GGA-TTGCCT3’) and an R-primer (5’~AAA-GTC-TGA-CAAACT-GAG-TCT-GCA-AC-3’). One /lg of total RNA, 1 mM each of dATP, dGTP, dCTP and dTTP, 20U of M-MLV RTase (RNase H-), 1OU of RNase inhibitor and the R primer (1.25 PM) were mixed in a final volume of 20 ~1. Reverse transcription was performed at 42°C for 30 min, and then the reaction mixture was heated at 99°C for 5 min. The reaction product was subjected to PCR in a lOO-~1 reaction solution containing 2.5 U of rTaq DNA polymerase, and 0.125 PM of each primer. After 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 2min, the reaction solution was analyzed by agarose gel electrophoresis. RESULTS
BY MAGTRATION TECHNOLOGY
501
section, magnetic separation is achieved during the sucking or dispensing of a suspension of magnetic particles by placing a magnet against the outside of the tip. Resuspension of the trapped magnetic particles is easily performed by sucking and dispensing the resuspension buffer with the magnet moved away from the tip. The inner diameter of the disposable tip for an intermediate region with a capacity of l-ml is only 2mm. Thus, the magnetic field in the tip is sufficiently strong to trap magnetic particles. The magnetic particle suspension passes through the high magnetic field region for a length of 15 mm, and almost all the magnetic particles are captured. Each sample is handled in the disposable tip. In the nucleic acid purification process, the buffer or reagent required is dispensed into the tubes first to reduce the possibility of cross-contamination between samples. Moreover, the separation and resuspension of magnetic particles are performed within the disposable tip. Thus, the risk of cross-contamination among the samples is greatly reduced and recovery is high compared with the conventional method. In addition, this method is amenable to automation and upgrading. Using Magtration Technology, two distinct instruments, SX-OG (Precision System Science, Matsudo, Chiba) and MFX-2000 (Toyobo), for the automation of nucleic acid extraction and purification were developed. The main components of the instruments are almost identical. SX-OG is controlled by a personal computer, and can be used for various purposes by changing protocols and parameters. MFX-2000 is designed especially for nucleic acid purification. Protocols for nucleic acid purification are preinstalled and the settings are optimized. Both instruments handle 24 samples successively. The instruments are composed of only two units, a stage on which to place the disposable tubes, tips and reagents, and the Magtration unit, a liquid dispensing unit with a magnet (Fig. 1). Thus, the system is very simple and inexpensive compared with other systems involving magnetic separation. Total DNA, RNA and plasNucleic acid extraction mids were extracted and purified by reagents developed for Magtration Technology based on the Boom method (Total DNA and RNA) (10) or by a glass-bead-based plasmid purification method (12). The basic procedure is outlined in Fig. 2. Cells or bacteria were lysed by
AND DISCUSSION
In MagtraMagtration Technology and instruments tion Technology, magnetic separation is performed in a specially designed disposable tip. The tip has three parts; a reservoir to aspirate the reaction mixture, a thin tip end, and an intermediate section. In the intermediate
FIG. 1. nology.
Tip and magnetic separation unit for magtration tech-
502
J. BIOSCI.BIOENG..
OBATA ET AL.
Sample FIG. 2.
Cell Lysis
Addition of magnetic particles
Release of nucleic acids
Recovery of nucleic acids
Schematic procedure for nucleic acid purification by magtration technology.
chaotropic reagents, causing the released nucleic acids which then absorbed on magnetic silica beads under high-ionic-strength conditions. By changing the ionic strength, each nucleic acid could be selectively captured. Unbound proteins, nucleic acids and other small cellular molecules were removed by washing with 70% ethanol. Bound nucleic acid could be eluted from the beads with Hz0 or low ionic strength buffer. This series of processes was complete within 10 min per sample. Extraction of total DNA from human whole blood The yield of genomic DNA from and HeLa cells human whole blood was 2 ~g/lOO ~1. The A26O/A280 ratio of the purified DNA was 1.8kO.l. The yield and purity were comparable to values for other commercially available DNA extraction kits (Fig. 3A). The extracted genomic DNA was subjected to PCR amplification. As shown in Fig. 3B, exon 5 and exon 8 of the human p53 gene were successfully amplified from the purified genomic DNA. The yield of genomic DNA from HeLa cells was 2.9
(A) 12
Magnetic capture of nucleic acids
(B) 123
pg/5 x lo5 cells and the A260/A280 ratio of the purified DNA was 1.84. Although the yield measured from the OD was significantly low compared with those obtained using other methods (8.1 to 9.7 ,ug), the yield estimated from the results of gel electrophoresis was not (data not shown). This result suggests that the purity of the DNA purified by this method is relatively high compared with that of DNA obtained using other methods. Purification of total RNA and amplification of cDNA encoding the transfer& receptor by RT-PCR Total RNA was purified from HeLa cells and HL60 cells. The yield and purity of the RNA purified from 2 x IO6 HL60 cells were 18 lug and 1.87 (A26O/A280), respectively. The values were comparable to those of manual extraction using the AGPC method (13 pg and 1.9 (A260IA280)). Figure 4A shows the results of agarose gel electrophoresis of the purified RNA. The 18s and 28s rRNAs were clearly observed. Total RNA extracted from 2 x 106 HeLa cells was used for RT-PCR targeting the transferrin receptor mRNA. The transferrin receptor gene was amplified only when reverse transcription was performed prior to PCR (Fig. 4B) Plasmid purification and restriction digestion E.
(4
(W 1
2
1
2
3
.Exon 8 (445 bp) 5 (310 bp)
FIG. 3. Purification of total genomic DNA and PCR amplification. (A) 1, Molecular weight markers 1 phage DNA digested by PstI; 2, total genomic DNA from a whole blood cell purified by magtration technology. (B) 1, Molecular weight markers 1 phage DNA digested by PstI; 2, PCR-amplified exon 5 of the ~53 gene from total genomic DNA of a whole blood cell purified by magtration technology; 3, PCR-amplified exon 8 of the p53 gene from total genomic DNA of a whole blood cell purified by magtration technology.
FIG. 4. Purification and RT-PCR of total RNA of the human transferrin receptor. (A) 1, Total RNA from HL60 cells purified manually by the AGPC method; 2, total RNA from HL 60 cells purified by magtration technology. (B) 1, Molecular weight markers, R phage DNA digested by ZfindIII; 2, RT-PCR product obtained with RTase; 3, RT-PCR product obtained without RTase.
VOL. 91, 2001
AUTOMATED NUCLEIC ACID PURIFICATION
BY MAGTRATION TECHNOLOGY
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no less than those for manually purified nucleic acids. The main shortcoming of the system is that it is timeconsuming to purify many samples. Since the instrument is equipped with only one nozzle, purification is individually performed. Thus, it takes about 4 h to purify nucleic acid from 24 samples. This problem could be easily overcome by developing an instrument equipped with multiple nozzles. We have already developed systems with 6, 12 and 96 nozzles. The 6-nozzle instrument reduced the time required for purification 6-fold, with 6 samples being treated in 10min. Nucleotide purification from 48 samples could be performed in only 80 min. REFERENCES
FIG. 5. Purification of plasmid by magtration technology and its restriction digestion. 1, Molecular weight markers, A phage DNA digested by iYindII1; 2, plasmids, pUCl8 purified by magtration technology; 3, KpnI-digested pUCl8 purified by magtration technology; 4, &a-I-digested pUCl8 purified by magtration technology.
JM109 harboring plasmid pUC18 was cultured in 1.2 ml of L-broth and used for plasmid purification by MFX2000. E. coli cells were lysed by alkaline/SDS. Following neutralization, the cell debris of genomic DNA, denatured protein and other polymers were captured with magnetic beads and removed by magnetic separation. Silica magnetic beads were then added to the clear lysate, and the plasmid was purified. The yield of plasmid from E. coli was 3 to 6 pg/1.2 ml of broth (JM109/pUC18) and the A260/A280 ratio was 1.8fO.l. There was almost no contamination by genomic DNA or RNA. The purified plasmid was completely digested by KpnI and Z&z1 (Fig. 5). The yield and purity of the extracted plasmid were no less than those of manually purified plasmid. The purified plasmid could be used directly for sequencing as a template. To confirm that no cross-contamination among samples occurs during nucleic acid purification by Magtration Technology, 100 ~1 of human serum containing the HCV virus at a concentration of 107/ml and the same volume of human serum without the virus were alternatively placed in 24 sample stands of the MFX2000, and nucleic acid purification was performed. The purified nucleic acid was eluted in Hz0 and subjected to HCV RNA detection by RT PCR using the Amplicore-HCV kit (Nippon Roche, Tokyo). All 12 nucleic acids purified from negative sera were judged to be HCV-RNA-negative while other nucleic acids obtained from positive sera gave positive signals (unpublished data). The present results clearly demonstrate that Magtration Technology enables automated purification of nucleic acids from various sources. The automation instrument performed with high efficiency in the purification of total genomic DNA, total RNA and plasmids. The yield and purity of the purified nucleic acids were coli
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