Journal of Bioscience and BioengineeringVOL. 110 No. 5, 608–613, 2010

www.elsevier.com/locate/jbiosc

Plasmid DNA of high quality purified by activated charcoal

Jae-Young Kim,1 Chunghee Cho,2 and Byung-Nam Cho1,3,⁎

⁎ CorrespondE-mail add

1389-1723/$doi:10.1016/

Research Center for Biopharmaceutical Lead Molecule, The Catholic University of Korea, Bucheon 420-743, Republic of Korea1 Department of Life Science,Gwangju Institute of Science and Technology (K-JIST), Gwangju 500-712, Republic of Korea2 and Department of Life Science, The Catholic University of

Korea, Bucheon 420-743, Republic of Korea3

Received 20 April 2010; accepted 22 June 2010Available online 16 July 2010

Demand for plasmid DNA of high purity and safety has increased with rapid advances in gene therapy and DNA vaccines inaddition to basic DNA study. Using activated charcoal (AC), we have developed protocols for pure plasmid DNA. Plasmid DNAextracted by the alkaline lysis method was inevitably contaminated with nucleotide fragments. Treatment with AC duringpurification instead of RNase completely removed nucleotide fragments in the final plasmid DNA and the removing capabilityof AC was dose dependent on AC quantity. Of note is that nucleotide fragments less than 0.4 kbp were effectively removed byAC and purification up to 500 ml was easily achieved. Taken together, inexpensive AC effectively removed the troublesomenucleotide fragments and practically substituted for expensive RNase. The resultant plasmid DNA has enough quality neededfor basic DNA study and application.

© 2010, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Plasmid DNA; Activated charcoal; Purity; Safety]

Plasmid DNA has been used as one of the most important tools inbasic and applied researches including molecular biology, medicine,and the biotechnology industry. Plasmid DNA is suitable for selection,subcloning, and amplification of the targeted genes with an idealselectivemarker in a host system (1,2). The amplified genes have beenused for subsequent experiments including gene sequencing, recom-bination, PCR, and transgene construction for transgenic organism(2,3). Recently, demand for plasmid DNA of high purity and safety hasincreased vastly in response to rapid advances in the field of geneticmedicine, gene therapy and DNA vaccines (4–6). Both viral and non-viral vectors can carry out the transport of the therapeutic genes to thenuclei of the target cells (7–12). In the case of nonviral approaches togene therapy, plasmid DNA has become a promising gene deliveryvector because it can easily be produced (2,3,12). And the risk ofunwanted immunogenic reaction (13) or oncogenic activation (14) isnegligible in comparison to viral vectors. Therefore, plasmid DNA ofhigh quality and large quantity (gram or kilogram amounts) isessential in basic research and clinical application (15).

Methods for plasmid DNA preparation included extraction orchromatography. Extraction system is a highly scalable unit operationand requires relatively inexpensive chemicals and equipments(16,17). By contrast, chromatography including anion exchange orligand affinity chromatography does have relatively high selectivitywhereas chromatography needs some improvement for large quan-tities (15,18). Recently, much improvement was done using variousforms of chromatography (19–21). The commercial kits available formini-, midi-, and maxi-preparation according to the volume of

ing author. Tel.: +82 2 2164 4358; fax: +82 2 2164 4765.ress: ocean@catholic.ac.kr (B.-N. Cho).

- see front matter © 2010, The Society for Biotechnology, Japan. Allj.jbiosc.2010.06.008

Escherichia coli culture have generally been based on the extractionmethod. However, these kits do not completely remove the nucleotidefragments including gDNA, and various RNAs in plasmid DNA. Theseparation of RNA from DNA is particularly challenging because of thesimilar physiochemical properties of both nucleic acids. To remove theRNA, the bovine RNase A has been generally used. However, animal-free production of plasmid DNA with low cost is important for basicresearch and clinical application (22) although recently developedrecombinant RNase Ba can be used (23). Other contaminations such asphenolic compounds, polysaccharides, and salts can originate fromhost cells or preparative reagents used for the extraction of plasmidDNA (24). Contaminating nucleotide fragments increase the error inestimating accurate quantity of plasmid DNA and they also inhibit PCRby influencing Taq DNA polymerase activity (25). Contaminated acidicpolysaccharides are potential inhibitors of restriction enzymesincluding HindIII (24). Salts generate ions and the ions disturbprotein-DNA bonds (25,26). These contaminations distort the resultsin many analytical applications and lead to incorrect interpretations.

AC has been used to remove various contaminants fromwater, soil,and air and has proved to have wide applications in daily life,pharmacy, microbiology, and industry (27–31). Characteristically, AChas many micro-pores in its surface that adsorb contaminations andremove them (28,32). In this study, the adsorptive capability of ACwas used to remove contaminations during the purification of thehigh-quality plasmid DNA.

MATERIALS AND METHODS

Plasmid DNA preparation and RNase treatment Plasmid DNA that containspOTB7-human inhibin alpha (MGC 12547, Invitrogen, Carlsbad, CA, USA) was

rights reserved.

FIG. 1. Diagramof alkaline lysis purificationwithAC. Purificationmethod of alkaline lysiswas described inmaterials andmethods. Alkaline lysiswithACusedAC instead of RNase. TE: Tris–EDTAbuffer, Ph: phenol, Ch: Chloroform, plasmid DNA: plasmid DNA,−R:−Removal, Sol I, II, III: solution I, II, and III. The numbers indicate each step of alkaline lysis method.

PLASMID DNA PURIFIED BY ACTIVATED CHARCOAL 609VOL. 110, 2010

purchased. The plasmid DNA is 3,184 bp. Preparation of the plasmid DNA was carriedout according to the procedure of Sambrook and Russell (Fig. 1) (1). Briefly, a singlecolony of transformed E. coli was set up a 500 ml overnight culture in 2000 ml plasticflask with chloramphenicol for plasmid amplification and the cells were harvested bycentrifugation at 8000g for 15 min at 4 °C. The bacterial pellet was resuspended in tubeof 10 ml of Solution I composed of 50 mM glucose, 10 mM EDTA pH 8.0, 25 mM Tris–ClpH 8.0, and 5 mg/ml lysozyme. Twentymilliliters of fresh Solution II composed of 0.2 MNaOH and 1% SDS was added into tube and mixed well by inverting gently 6 times andthe tube was incubated on ice for 10 min. Fifteen milliliters of ice-cold Solution IIIcomposed of 3 mM potassium acetate and glacial acetic acid was added into tube andmixed very gently and the tube was incubated on ice for 10 min. The sample solutionwas centrifuged at 8000g for 30 min at 4 °C and the supernatant was carefullytransferred into the new tube together with 0.6 volume of isopropanol. The tube wasinverted several times, stored for 30 min at 4 °C, centrifuged, and the supernatant wasdiscarded. The pellets were washed with 100% ethanol and then 70% ethanol. Afterdrying, the pellet was resuspended with TE buffer. Treatment with bovine RNase(Ribonuclease A, USB, Cleveland, OH, USA) was carried out and after the RNasetreatment, phenol/chloroform (1:1) extraction was performed and the supernatantswere removed after centrifugation at 12,000g for 5 min. One tenth volume of 3 Msodium acetate and 2 volumes of ethanol were added to the supernatant in a tube, andthe tube put in ice for 30 min. The precipitated solution was centrifuged at 12,000g for10 min at 4°C, and the pellet was washed with 70% ethanol. The pellet was dried andresuspended in TE buffer. Then, the amount of plasmid DNAwas measured by OD260nm.

To examine the effect of RNase A on removing the nucleotide fragments, plasmidDNA purified without RNase treatment was used. One or five micrograms of theplasmid DNAs was treated with 0.1 μg of RNase for 30 min at 37 °C, electrophoresed,and stained with EtBr.

Removal of nucleotide fragments by AC To remove the nucleotide fragments,AC treatment was performed. In case of AC treatment, RNase treatment was omittedwith the same protocol of alkaline lysis method. The amounts of plasmid DNA obtainedwithout RNase treatment were temporarily estimated by OD260nm after TE addition.Then AC was added into the tube and vortex was immediately followed at roomtemperature. The pure plasmid DNA in supernatant was obtained after centrifugationfor 5 min at 6000 rpm and final quantification was done using OD260nm.

To check the adsorptive capability of AC, 5 mg of AC (Sigma, St. Louis, MO, USA) wasput into the tubes which contained ink (Parker, Newhaven, East Sussex, UK) plusdistilled water (DW) (10 μl of ink plus 990 μl of DW), ink plus ethanol (10 μl of ink plus445 μl of DW and 445 μl of 100% ethanol), and ink plus TE buffer (10 μl of ink plus 990 μlof TE), respectively. No addition of AC was done in control which contained ink plusDW. The tubes were then incubated for 10 min and the tubes except control were then

centrifuged at 5000g for 5 min to remove the ink-absorbed AC. The supernatants weretaken and photographed.

The adsorptive capability of AC was first applied to remove the nucleotidefragments: 5 mg of AC was put into the tubes of 1, 2, 4, and 8 μg of plasmid DNAspurified with above method without RNase treatment, respectively. Working AC wasprepared by adding of AC (1 mg) into DW (10 μl).

To determine dose dependent effects of AC on removing the nucleotide fragments,plasmid DNAs which were isolated from 1.5 ml of E. coli culture were treated with 0,1.25, 2.5 and 5 mg of AC, respectively. In case of treatment of AC (5 mg) with one time,nonspecific loss of plasmid DNA was expected. To evade this, divided AC with the sametotal 5 mg AC was put into tube.

In the next step, we determined the differential removing capability of ACdepending on DNA size. To this, DNA ladder (Promega, Madison, WI, USA) instead ofplasmid DNA was used during AC treatment.

To verify purity and precise quantification of plasmid DNA, plasmid DNA isolatedfrom 1.5 ml of E. coli culture was treated with 5 mg of AC and quantitative analysis ofthe plasmid DNA was carried out with Image Analyzer (Kodak Image Station 4000,Kodak, Rochester, NY, USA) after electrophoresis. For temporary quantification forloading and subsidiary quantification, OD260nm was measured whereas ratio ofOD260nm/OD280nm was obtained for the purity check.

Enzyme reaction and transformation of plasmid DNA In order to check goodquality of plasmid DNA, we investigated reaction of restriction enzyme on plasmidDNA. Five micrograms of plasmid DNA purified with AC instead of RNase was mixed 0.5μl of EcoRI (Promega, Madison, WI, USA) in a reaction buffer (1 μl BSA, 1 μl 10× reactionbuffer) and incubated at 37 °C for 10 min. After chloroform extraction without phenol,enzyme-treated plasmid DNA was electrophoresized and stained with EtBr. Anothercheck for good quality was done by transformation. Transformation of the plasmid DNAwas carried out according to the procedure of Sambrook and Russell (1). Briefly, sterilesolution of 10 mM MOPS (morpholinopropane sulfonic acid, pH 7.0) and 10 mM RbClwas added into the tube containing E coli. After centrifugation at 4000g for 10 min at4 °C, the cell pellet was resuspended in 1 ml of solution of 0.1 MMOPS (pH 6.5), 50 mMCaCl2, and 10 mM RbCl. After 15 min incubation on ice, another centrifugation wasdone. The pellet was again resuspended in 0.2 ml of 0.1 MMOPS (pH6.5), 50 mM CaCl2,and 10 mM RbCl. And tube was filled with 3 μl of DMSO (dimethylsulfoxide) and 1 μg ofplasmid DNA in a volume of 10 μl. After 30 min incubation, the tube was transferred to awater bath at 42 °C for 30 s. And the tube was filled with 1 ml of LB medium and wasincubated for 60 min without shaking. After centrifugation, supernatant was discardedand the pellet was resuspended in 100 μl of LB medium. Finally, the transformedcompetent cells were transferred onto agar LB medium with ampicillin and the plateswere incubated at 37 °C for 12–16 h.

FIG. 3. Adsorptive capability of AC. (A) Photograph after AC treatment on ink. After ACaddition, the tubes were centrifuged to remove AC at 30 min later. The new tubestransferred with supernatant were photographed. No addition of AC was done incontrol. Cont (control): 10 μl of ink+990 μl of DW, DW: 5 mg of AC+10 μl of ink+990μl of DW, DW/EtOH (ethanol): 5 mg of AC +10 μl of ink +445 μl of DW +445 μl ofEtOH, TE: 5 mg of AC +10 μl of ink +990 μl of TE buffer. (B) The plasmid DNA wasobtained without RNase treatment and the amount was estimated by OD260nm after TEaddition. The plasmid DNA after AC treatment (5 mg) was electrophoresed, stained,and photographed. M: λ DNA/HindIII marker, −AC: no AC treatment, +AC: ACtreatment, plasmid DNA: plasmid DNA.

610 KIM ET AL. J. BIOSCI. BIOENG.,

Scale-up purification of plasmid DNA To meet the need for large quantity, weinitially applied AC treatment to 500 ml of E. coli culture which is a maximum level in alaboratory scale. Plasmid DNA was obtained according to the alkaline lysis methodwithout RNase treatment. ACs as powder in proportion to the plasmid DNA extractwhich come from E. coli culture ranging from 62 ml to 500 ml in volume (AC: E. coliculture volume=5 mg/1.5 ml) were put into the tubes which contained plasmid DNAextract.

RESULTS

Incomplete removal of nucleotide fragments with RNase Ac-cording to the alkaline lysis method (Fig. 1), treatment with RNaseduring plasmid purification removed nucleotide fragments in the finalplasmid DNA. However, some nucleotide fragments still remained inspite of RNase treatment as revealed in lanes marked as+RNase(Fig. 2).

Effective removal of nucleotide fragments by AC Initially, weexamined the adsorptive capability of AC. To visualize the adsorptivecapability, 5 mg of AC was added into the ink solution mixed withdistilled water (DW), ethanol, and TE, respectively. The result wasthat AC completely eliminated the ink particle in DW and TE, butpartially in ethanol (Fig. 3A), suggesting that the adsorptive capabilityis influenced by the surrounding conditions. This adsorptive capabilityof AC was then used to remove the contaminants, especially smallnucleotide fragments. When 5 mg of AC was added into the tubes of 1,2, 4, and 8 μg of plasmid DNAswhich had not been treatedwith RNase,the nucleotide fragments were completely removed as revealed in allAC-treated groups (Fig. 3B). Thus adsorptive capability can be usedeffectively to remove the nucleotide fragments which are inevitablygenerated during purification process. The removing capability of ACwas investigated in more detail. First, we determined the dose effectof AC on removing the nucleotide fragments. When 1, 1.25, 2.5, and5 mg of AC were added into the tubes containing plasmid DNAextracted from 1.5 ml (wet mass=11 mg) of E. coli culture, thenucleotide fragments were removed in proportion to the amount ofAC treated. Of note is that 5 mg of AC was enough to remove thenucleotide fragments (Fig. 4A). Next, we investigated possiblenonspecific loss of plasmid DNA when 5 mg of AC was treated at atime. To this end, repeated treatment with divided AC without changein a total amount was adopted. When 5 mg of AC with single (5 mg),twice (2.5 mg), and four (1.25 mg) times were treated, respectively,no change in the amount of the recovered plasmid DNA in each tubewas observed (Fig. 4B), suggesting that there is no nonspecific loss

FIG. 2. Incomplete removal of nucleotide fragments by RNase treatment. The plasmidDNA was obtained without RNase treatment and the amount was temporarilyestimated by OD260nm measurement after TE addition. Then, one or five microgramsof plasmid DNA were treated with 0.1 μg of RNase. M: λ DNA/HindIII marker, DNA:pOTB7 plasmid DNA, −RNase: no RNase treatment, +RNase: RNase treatment, nf:nucleotide fragment, plasmid DNA: plasmid DNA.

other than adsorptive capability. Last, we investigated the sizelimitation of the nucleotide fragments during elimination process.When linearized DNA ladder with known size was used since thefragmented nucleotides appeared to generated by fragmentation ofgenomic DNA and various RNAs, DNA fragments less than 0.4 kbpwere removed by addition of AC (Fig. 5). Thus, elimination ofnucleotide fragments by AC was very selective in size.

Pure plasmid DNA with good yield The selective removal ofnucleotide fragments during purification of plasmid DNA made itpossible to precisely quantify the plasmid DNA. When we observedthe band of the plasmid DNA using Image Analyzer after AC treatmentand electrophoresis, no change of the amount of plasmid DNA wasfound between before and after AC treatment (Fig. 6A, upper bandrepresented as plasmid). When we quantified the band intensity withthe help of Molecular Software (Version 4.5, Kodak, Rochester, NY,USA), total amounts of nucleic acids containing plasmid DNA andnucleotide fragments were about 60 μg in control and 40 μg in ACtreated group, respectively (Fig. 6B). The recovered plasmid DNA incontrol and AC treated group was almost the same (40 μg) (Fig. 6C)whereas the removing amount of nucleotide fragments was about 20in AC treated group (Fig. 6D). Thus, the yield of plasmid DNA wasabout 40 μg/1.5 ml of E. coli culture according to the result of Imageanalysis and independent quantification using OD260nm. This precisequantification is very important because the exact amount of plasmidDNA is a starting point in subsequent experiments using plasmid DNA.The plasmid quality was analyzed by examining the gel under UV lightand measuring its ratio of OD260nm : OD280nm. The ratio was about2.19. High-purity plasmid DNA is important in experiments includingsubcloning, recombination, and production of the transgenic organ-ism for basic and applied researches.

Enzyme reaction and transformation of plasmid DNA Thequality of purified plasmid was expected to be suitability for enzyme

FIG. 4. Removing capability of AC (A) and no nonspecific loss of the plasmid DNA (B).(A) The plasmid DNA obtained from 1.5 ml of E. coli culture was treated with indicateddoses of AC. Then the plasmid DNA was electrophoresed, stained, and photographed.(B) The plasmid DNAs were treated with AC (total 5 mg) with single (5 mg/each time),twice (2.5 mg/each time), and four (1.25 mg/each time) times, respectively, asindicated. M: λ DNA/HindIII marker, +AC: AC treatment, −AC: no AC treatment, nf:nucleotide fragment.

FIG. 5. Differential removing capability of AC on DNA size. AC was put into the tubewhich contained DNA ladder of 100 bp instead of plasmid DNA extract. Aftercentrifugation, DNA ladder was electrophoresed, stained, and photographed.

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reaction and transformation. When the plasmid DNA was reactedwith an EcoRI enzyme, the cleavagewasmore efficient in the presenceof AC (+EcoRI plus+AC) than in the absence (+EcoRI plus −AC)(Fig. 7A). Thus, plasmid DNA obtained using AC was suitable forenzyme reaction. When plasmid DNA was transformed into compe-tent cell, successful transformation was observed (Fig. 7B). Thus,plasmid DNA obtained using AC was also suitable for transformation.

Scale-up purification of plasmid DNA For upcoming demandfor gene therapy and DNA vaccines, we applied AC treatment to thescale-up preparation. When we added various amounts of AC inproportion to the E. coli culture ranging from 62 ml (wetmass=0.45 g) to 500 ml (wet mass=3.67 g) in volume, thenucleotide fragments were successfully removed (Fig. 8).

DISCUSSION

Plasmid DNA is at the center of bioscience and the biotechnologyindustry as mentioned in the introduction. During purification,however, impurities such as nucleotide fragments, cellular compo-nents, and ions which had originated from the isolation process couldnot be avoided. These impurities inhibited precise quantification ofplasmid DNA and subsequent experiments. Thus, these impuritiesmust be removed before using plasmid DNA. In order to remove theseimpurities we used the adsorptive capability of AC that strengthensthe surface porosity of a charcoal.

As to the purity of plasmid DNA, remaining nucleotide fragmentsas well as other impurities have been problems for subsequentstudies involving recombination, transgene construction for trans-genic animal production and molecular biology. The purity is alsoexpected to be a central issue for upcoming gene therapy and DNAvaccines. As observed in Fig. 2, nucleotide fragments in the plasmidextract which presumably come from broken genomic DNA,plasmid DNA, and RNAs could not be completely removed duringalkaline lysis purification. To solve this problem, RNase has beenused in alkaline lysis method. As to the removal of RNA, twomethods are available. The first method is to destroy RNA withRNase and remove destroyed RNA as adopted in alkaline lysismethod. The second method is to separate RNA from DNA withoutdestroying. These methods include size exclusion chromatography(SEC), aqueous two-phase system, high salt precipitation, andpolycation-using method (15–22). As an alternative method for thefirst method, we tested AC in place of RNase. The result was that ACremoved completely nucleotide fragments with simple treatment ina dose dependent manner (Fig. 3B). The 5 mg of AC was enough toremove the nucleotide fragments for 1.5 ml of E. coli culture whichis an amount for commercial minipreparation kit.

As to the safety of plasmid DNA for gene therapy and DNAvaccines, the animal-originmaterials must be avoided (23). The safetyis more important than purity in the plasmid DNA for clinicalapplications. In this aspect, AC is an excellent substitute for bovineRNase which has been used for plasmid purification after obtainingfrom animal. AC successfully and efficiently removed the nucleotidefragments (Fig. 4). Recently developed recombinant RNase can be asubstitute, but is expensive for use (23). For the improvement ofsafety, AC can apply anytime to remove the doubted impurities in theplasmid DNA prepared by other methods. Thus AC can be used forsafety improvement in the basic step of plasmid DNA purification or inthe supplementary step in other method.

As to the selectiveness of removing unwanted nucleotidefragments, AC was excellent. Of note is that AC removed thenucleotide fragments under 0.4 kbp (Fig. 5). This selectiveness canpreserve plasmid DNA intact during purification which is usually over3.0 kbp in size. Thus this selectiveness allows wide application ofplasmid DNA purification regardless of the various kinds of plasmidvectors.

FIG. 6. Complete removal of nucleotide fragments by AC (A) and precise quantification (B, C, D). (A) Five microliters of plasmid DNA (Total 50 μl of 1.5 ml E Coli culture) obtainedwithout AC treatment (lanes 1–6) and with AC treatment (lanes 7–12) after no RNase treatment in 6 independent purifications were loaded, electrophoresed, stained, andphotographed. Total amounts of nucleic acid (DNA+RNA) (B), plasmid DNA (C), and nucleotide fragments (D) were calculated by quantification analysis with Image Analyzer. M: λDNA/HindIII marker, −AC: no AC treatment, +AC: AC treatment, NF: Nucleotide fragment, (n=6, mean±SE, **; pb0.01).

612 KIM ET AL. J. BIOSCI. BIOENG.,

In addition to the removal of nucleotide fragments, we testedindirectly whether AC could remove ions and other impurity. It wasknown that ions originated from chaotropic salts increase electro-static free energy and inhibit the stability of a protein-DNA complex(25,26) and acidic polysaccharide as a component of cellularmembrane is a potential inhibitor of restriction enzyme (24).Evidently, the impurities including nucleotide fragments inhibitedthe normal reaction of restriction enzyme as seen in lane representedas+EcoRI plus −AC (Fig. 7A). However, treatment with AC resulted

FIG. 7. Suitability for enzyme reaction (A) and transformation (B). (A) All plasmid DNAswere purified without RNase treatment. Intact: plasmid DNA was not reacted withEcoRI with no AC treatment, +EcoRI/−AC: plasmid DNA was reacted with EcoRI withno AC treatment, +EcoRI/+AC: plasmid DNA was reacted with EcoRI after ACtreatment, nf: nucleotide fragment. (B) Purified plasmid DNA was transformed asdescribed in materials and methods. −AC: without AC treatment, +AC: with ACtreatment.

in enhanced activities of the restriction enzyme as seen in lanerepresented as+EcoRI plus+AC, suggesting that other impuritieswere also removed and resulted in good quality of plasmid DNA(Fig. 7A). Another indirect evidence for good quality of plasmid DNAwas the transformation result as revealed in Fig. 7B.

As to the yield and scale-up of plasmid DNA preparation, ourmethod is effective. The yield of plasmid DNA was about 40 μg/1.5 mlof E. coli culture compared to the 7.5–90.0 μg/1.5 ml in other studies(1–3,33) (Fig. 6). Though direct comparison is not appropriate sincethe plasmid number is dependent on the copy number of plasmidDNA, our yield seemed be normal. The scale-up purification of plasmidDNA is important since large amount ranging from gram to kilogramfor gene therapy has been expected. Treatment of AC up to 500 ml ofE. coli culture was not difficult to carry out (Fig. 8). If we divide 500 mlas a unit for the larger E. coli culture volume, we can easily handle it.And more convenient treatment with AC which does not usecentrifugation is under investigation.

FIG. 8. Scale-up purification of plasmid DNA. Treatment of AC was basically done asdescribed in materials andmethods. ACwas put into the plasmid DNA extracted from E.coli culture (AC weight: E. coli culture volume=5 mg: 1.5 ml). E. coli culture volumeranged 62 ml (wet mass=0.45 g) to 500 ml (wet mass=3.67 g). The plasmid DNAequal to amount from 0.15 ml (wet mass=11 mg) of E. coli culture was electro-phoresed, stained, and photographed. M: λ DNA/HindIII marker.

PLASMID DNA PURIFIED BY ACTIVATED CHARCOAL 613VOL. 110, 2010

As to the endotoxin, AC treatment in our study could not removethe endotoxin. However, simple treatment of butanol in plasmid DNAextract was enough to remove the endotoxin (data not shown).

In summary, AC treatment in place of RNase has severaladvantages. Firstly, AC is not an animal origin compared to bovineRNase, which is critical, especially in gene therapy and DNA vaccines(4–11,34). Thus the plasmid DNA purified with AC is safe. Secondly,AC is much cheaper in price than bovine or recombinant RNase, savingthe cost especially in large scale preparation. Thirdly, AC can be usedfor a long timewithout change. Thus, AC can be used conveniently andeffectively in place of RNase to remove the nucleotide as well as otherimpurities. Finally, AC can be used anytime to remove the doubtedimpurities in plasmid DNA prepared by other methods. The resultantplasmid DNA has enough quality needed for basic DNA study andapplication including gene therapy and DNA vaccine.

ACKNOWLEDGEMENTS

We thank Jun-Hyuk Kim and Sung Jae Lee for their technicalsupport. This work was supported by a grant from the "GRRC" Projectof Gyeonggi Provincial Government (to B.N. Cho), Republic of Korea.

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