Anal. Chem. IQGa, 65, 1323-1328
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Rapid Purification of Double-Stranded DNA by Triple-Helix-Mediated Affinity Capture Huamin Ji and Lloyd M. Smith' Department of Chemistry, University of Wisconsin-Madison,
Madison, Wisconsin 53706
A simple and rapid method for the preparation of highly pure plasmid DNA has been developed.The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidincoated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUCl9 DNA by insertion of a 40-basesequence suitable for triplehelix formation. The approach was also found suitable for the purification of X bacteriophage DNA.
two most successful motifs employed to date are the use of specific DNA-binding proteins and the use of triple-helix DNA. Triple-helix DNA, in particular, has proven to be a powerful and fairly general approach to DNA targeting. It is based upon the specific binding of pyrimidine oligonucleotides to the purine strand in duplex DNA, forming a local triplehelical s t r ~ c t u r e . The ~ , ~ pyrimidine oligonucleotide binds in the major groove of DNA parallel to the purine WatsonCrick strand through the formation of Hoogsteen hydrogen bonds. Specificity is derived from thymine (T) recognition of adenine-thymine (AT) base pairs (TaAT triplets) and protonated cytosine (C+) recognitionof g u a n i n e c y h i n e (GC) base pairs (C+.GC triplets). Triple-helix complexes have proven useful for site cleavage,5,7-10for inhibition of sequencespecific binding proteins, and as a promising antisense DNA strategy.6J1-16Recent papers have demonstrated the potential of triple-helix-mediated capture for the enrichment and screening of recombinant DNA libraries17J8 as well as for the isolation of PCR products.19 In this paper, a method is described for the triple-helixmediated affinity capture (TAC purification) of plasmid DNAs from a bacterial cell lysate. A modified cloning vector was constructed containing the triple-helix-forming region, and affinity capture of the vector containing a cloned insert is achieved by use of a biotinylated triple-helix-forming capture oligonucleotide in conjunction with streptavidincoated magnetic beads. Microgram quantities of high-purity plasmid DNA are readily obtained from small (1.5 mL) bacterial cultures, with no detectable contamination from RNA or chromosomal DNA. The approach has also been used with similar results for the affinity purification of X bacteriophage DNA.
INTRODUCTION The preparation of DNA plays an important part in virtually all work in molecular biology. The DNAs needed very widely in their nature, from small single-stranded DNAs such as M13 templates for DNA sequence analysis to intact yeast artificial chromosomes (YACs),entire human chromosomes, or total genomic DNA. Virturally all methods presently employed for DNA purification employ classic procedures such as extraction and centrifugation, which tend to be rather labor-intensive and do not lend themselves readily to automation. This poses a problem for large-scale projects such as the Human Genome Initiative, for which scale-up and automation are critical to the success of the project.lV2 In considering ways to address this problem, one attractive approach is to employ affinity methods. In principle, if one could target a DNA-binding agent to a desired region of DNA, it would be possible to affinity-purify the desired target molecule directly from a complex mixture. The target molecule could be immobilized on a support particle, separated from other solution components by appropriate washing steps, and eluted from the support to yield pure material. Such methods have been widely used for a variety of applications3and are relatively straightforward to automate.4 There has been substantial interest recently in this general problem of sequence-specific DNA targeting. Much of the driving force for this interest has stemmed from the desire to developgeneral methods for the directed cleavage of doublestranded DNA targets (artificial restriction enzymes), as well as for gene therapy and pharmaceutical applications. The (1)Hood, L.E.; Hunkapiller, M. W.; Smith, L. M. Genomics 1987,1, 201-212. (2)Smith, L. M.; Hood, L. E. Bio/Technology 1987,5,933-939. (3)Fox, T.0.; Savakis, C. In Solid Phase Biochemistry, Analytical and Synthetic Aspects; Schouten, W. H., Ed.; John Wiley & Sons: New York, 1983;pp 189-221. (4)Fry, G.;Lachenmeier, E.; Mayrand, E.; Giusti, B.; Fisher, J.; Johnson-Dow, L.; Cathcart, R.; Finne, E.; Kilaas, L. BioTechniques 1992, 13,124-131. 0003-2700/93/0365-1323$04.00/0
EXPERIMENTAL SECTION Oligonucleotides. All oligonucleotides were synthesized by the University of Wisconsin Biotechnology Center on an Applied (5)Moser, H. E.;Dervan, P. B. Science 1987,238,645-650. (6)Helene, C.; Toulme, J.-J. Biochim. Biophys. Acta 1990,1049,99125. (7)Pei, D.; Corey, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1990,87,985&9862. (8)Strobel, S. A.; Dervan, P. B. Science 1990,249,73-75. (9)Strobel, S.A.; Doucette-stamm, L. A.; Riba, L.; Housman, D. E.; Dervan, P.B. Science 1991,254,1639-1642. (10)Povsic, T.J.; Strobel, S. A.; Dervan, P. B. J.Am. Chem. SOC.1992, 114,5934-5941. (11)Maher, L. J.; Wold, B.; Dervan, P. B. Science 1989,245,725-730. (12)Shin, J. A.m; Ebright, R. H.; Dervan, P. B. Nucleic Acids Res. 1991,19,5233-5236. (13)Maher, I. L. J.; Dervan, P. B.; Wold, B. Biochemistry 1992,31, 7C-81. (14)Duval-Valentin, G.; Thuong, N. T.; Helene, C. Proc. Natl. Acad. Sci. U.S.A. 1992,89,504-508. (15)Gee, J. E.; Blume, S.; Snyder, R. C.; Ray, R.; Miller, D. M. J.Biol. Chem. 1992,267,11163-11167. (16)Blume, S. W.; Gee, J. E.; Shrestha, K.; Miller, D. Nucleic Acids Res. 1992,20,1777-1784. (17)Ito, T.;Smith, C. L.; Cantor, C. R. Proc. Natl. Acad. Sci.U.S.A. 1992,89,495-498. (18)Ito, T.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1992,20, 3524. (19)Vary, C. P.H. Clin. Chem. 1992,38,687-694. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 into a unique NdeI site in the pUC19 vector. pUC19 vector DNA (New .~ Eneland Biolabs., Inc..~,Beverlv... MA). waa dieested completely with Ndel (all enzymes were purchased from New England Riolabs, Inc.!. Twooligonucleotides I5'-TAC l T A ACT CCTTCT TC'I'CCI'TTC TC'r TCTTTC CTT CTT TCTCTC TT(X'andY-TAGAAG AGACAA AGAAGG AAAGAAGAG AAA GGA GAA GAA GGA CTT AAG-3'. also shown in Figure 1 ) were annealed and ligated with the restricted vector. The ligation product wns digested again with Ndel to reduce the barkground of nonreromliinant transformants, transformed into Escherichia coli atrain DHhF'. and plated hy standard procedures." Thestructureof the resultant vector, ralled pHJ19, was run firmed hy restriction analysis and DNA sequencing acruw the insert sequence. which also permitted determination ofthe insert orientation. The pH.119 vector is 2732 bp in length and retains all t he features ofthe pllC I9 vector (ampicillin resistance, blue-whitecoloruelection. Qo~y~inkersitesJexcept that the original Ndel site was destroyed nnd B new AfIII site was created in its place. Prior to the construction of pHJ19. a similar vector denoted pUCI9A was constructed by insertion ofthe above triple-helixforming sequence between the BamHl and Xbol sites in the polylinker ofpUC19. This triple-helix vector was inserted into Apt10 as descrihed below, thereby introducing the triple-helixforming sequence into the hacteriophage. Subcloningof XDNA Hindlll and h DNA HaellI Digests into pHJ19Vcctor. Inorder torest pH.119,aswellastoobtain a numherofpHJ19clones containing inserts ofvaryinglengths. A DNA was digested with Hindlll or Hoe111 and cloned into pHJ19. ThepH.119DNA wasdigestedcompletely withHindlll or HinclI and dephosphorylated with calf intestinal alkaline phnsphatase. TheHindlll-digestedanddephosphorylatedvector DNA was ligated with the A DNA Hindlll fragments and transformed ns described ahove. The A DNA IIaelIl fragments were frartionated by size (toeliminateverysmall fragments from the ligation reaction) on a packed column of Sephacryl S-500 (Pharmacia LKB, Piscataway, NJi size exclwion gel matrix as descrihed elsewhere:' The fractionated fragments were ligated to the Hincll restricted and dephosphorylated pHJ19 and transformed asabove. White recombinant colonies were picked at random and grown in 2x TY broth rontaining 50 rrg mL ampicillin. The DNAs were prepared using TAC purification (Table 11. The purified DNAs rontaining Hindlll fragments wcredigested with Hind111toexcise the inserts. which were then sized hy gel electrophoresis. DNA Sequencing. DNA sequence analysis was performed with a Taq cycle sequencing kit (United States Hiwhemical. Cleveland, OH) acrording to the manufacturer's protocol. Purified dsDNA solution 15 rrL, -0.1 ug) was employed. [a-"-PldATP (>400 Ci mmol, 10 mCi mL; Amersham, Arlington Heights, 1L) was used lo label sequence products by primer extension for 4&50 cycles. Cloning of pUCI9A DNA in A@ LO Phage Vector. The pUCl9A DNA was linearized by digestion with EcoRI. ligated with EcoRI-digested A g t 10 vectorarms (Promega,Madison, WI), parkaged,and infected intothe E. coli strainC600HFlaccording to the manufacturer's protocol. The resultant phage DNA (denoted XgtIUT) was prepared and purified using a A DNA isolation kit from Qiagen. Triple-Helix-Mediated Affinity Capture (TAC) of Plasmid DNA. A detailed protocol is shown in Tahle I. Briefly, the pelleted bacterial cells are lysed by addition of 0.2 M NaOH and l r ; sodium dodecyl sulfate (SDS) and then neutralized with a solution of 3 M KOAc, pH 5.0. TriplexBeads are added and the solutionisincuhatedat roomtemperature for 15min. The beads are separated using the magnetic stand and washed sequentially with BB and WH. The DNA is eluted from beads with a 10-min incuharion in EB. For control experiments, plasmid DNAs were also isolated by conventional methods, as follows. The cell lysate was mixed ~~
~~
~
mure 1. Map of the vector pK119. Biosystems 380B DNA synthesizer (Foster City, CA). Biotin was incorporated at the 5' end by standard synthesis procedures usingbiotin phosphoramidite (Glen Research, Sterling,VA). The oligonucleotides were purified hy reversed-phase high-performance liquid chromatography or using a Sep-Pak C18 cartridge (Waters, Milford, MA) and concentrations were determined by spectrophotometry. The following three biotinylated oligonucleotides, 20,25, and 37 nucleotidesinlength, respectively, were prepared, havingheen designed to form a triple-helical complex with a target sequence incorporated intothepHJ19vectordescribed below: (1)5'-hiotinACT ACT CTT CTC TCT TTC TT-3'; (2) 5'-biotin-ACT ACT CTT CTC TCT TTC TTC CTT T-3'; (3) 5'-hiotin-ACT ACT CTTCTCTCTTTCTTC TCCCTTTCTTCTCTTTCCT-3'. All cytosines in the ahove oligonucleotides were 5-methylcytwines, which have been found to increase the stability of the Hoogsteen-paired triple-helix strand.20 Unless otherwise specified, oligonucleotide 2 was employed in all experiments. Buffer Solutions. The buffer solutions employed were as follows: Phosphate-buffered saline (PBS) 0.15M NaCl and 10 mM sodium phosphate, pH 7.5; binding buffer (BB) 1 M potassium acetate (KOAc) pH 5.0; elution buffer (EB) 50 mM Tris-HCI, pH 9.0; wash buffer (WB) 10mM NaOAc, pH 5.8,lOO mM MgCI2. Magnetic Beads. Streptavidin-coated magnetic beads (Dynabeads M-280, Dynal, Inc., Great Neck, NY) were utilized as a solid support. A magnetic separation stand (Promega, Madison, WI) was used to immobilize the heads during the supernatant removaland washsteps. The heads wereprewashed with binding buffer twice and resuspended in the same binding buffer. These heads were reoorted hv the manufacturer to Dossess 0.3 Dmol of biotin-binding sitesir; of heads. TriplexBeads. Magnetic beads (100 rL, 1mg of beads) are washed twice with PBS. 20 omol of biotinvlated caoture oligonurleotide in added, and t h e mixture is- kept at inom temperature for 15 min. The supernatant solution is removed, and the heads are washed twice sequentially with PRS, EH, and RR solutions. The resultant preparation of beads containing immubilized triple-helix oligonucleotide (henceforth referred to as TriplexBeads) are stable in PBS at 4 "C for at least 2 months without ohservahle degradation. This preparation procedure can bescaled upifdesired by proportionateincreasesin theamounts of magnetic beads and biotinylated oligonucleotide employed. The TriplexBeads can he reused multiple times with no degradation in performance. To regenerate the beads after use, they are washed tuice with 1.0 M 'his-HCI, pH 9.0, BB, and PHS sequentially and stored at 4 "C. pRJ19Vcctor. AderivativeofpUC19. pHJ19 (see Figure I). was constructed in which a triple-helix-formingsite was inserted (20) Hausheer.F. H.:ChandrsSingh.II.;Sair.J. D.;Flury.J. P.;Tufto. K. B.J . Am. Chem. SOC.1992, 114. 5356-5362.
~
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(211 Samhrook, .I.: Frihrh. E.F.; Manratis, T. Molerulor C l o n m ~A. 1.ohorohq Monuol: Cold Spring Harhor Laboratory Prms: Cold Spring Harbor. 1989.
Q2, Fit,gprald, M.C.; Skuwon, P.; Van Etwn, .I. L.: Smith. L. M.: Mead, D.A. Nurlvir Acids U p s . 1992.20.3753.-3162.
ANALYTICAL CHEMISTRY. VOL. 65. NO. 10. MAY 15. 1993 1925 Table I. Protocol for TriplexBeads Preparation and TAC Purifioation
Spin 1.5 mL of overnight culture in microcentrifuge for 20 s. Decant supernatant 2. Resuspend cell pellet in 100 p L of GET solution (50mM glucose, 10 mM EDTA, 25 mM Tris-HCI. DH8.0):vortex to 1.
dissolve completely 3. Add 200 pL of 0.2 M NaOH/l% SDS solution; invert to mix. Incubate on ice for 5 min 4. Add 150 pL of cold 3 M KOAc (pH 5.0) solution: gently invert to mix. Keep on ice for 5 min 5. Spin in microcentrifuge for 5 min. Transfer supernatant to a fresh tube 6. To bind, add 100 p L of TriplexBeads. Leave at room temper-
-506,517
ature for 15 min 7. To wash, immobilize beads with a magnetic stand and wash once with 100 pL of BE (1.0 M KOAc, pH 5.0) and once with 100 p L of WB (10 mM NaOAc, pH 5.8,100 mM MgCI2). Care-
fully pipet out all the liquid 8. To elute, add 50 p L of EB (50 mM Tris-HCI, pH 9.0). Leave at room temperature for 10 min 9. Immobilize the beads to the side wall of the tube; transfer the
supernatant to a fresh tube Making of TriplexBeads 1. Wash 1mL (10 mg of beads) twice with PBS (10 mM NaZHPO,, pH 7.5,0.15 M NaCI). Resuspend in 1mL of PES 2. Add 200 pmol of capture oligonucleotide. Incubate at roam temperature 30 min 3. Wash the beads twice with PES, twice with EB, and twice with BB. Keep in PBS at 4 "C until use BW-Qm-
Flgure 3. Agarose gel electrophoresis of plasmld DNAs purllid fmm cell lysates: (lane 1) pUCl9 DNA purified using TriplexBeads:(lane 2) pKJ19 DNA purified with streptavidlncoated magnetic beads but no added biotinyiated capture oligonudeotlde: (lane3 ) p W l 9DNA puriRsd by(a)fwmingthebipieheiixcomplex insoititonand(b)thenimmobiiWng the preformed complex to the streptavidin beads (see text): (lane 4) pKJ19 DNA purified with TriplexBeads: (lane 5) pKJ 19 DNA purlfled by phenollchioroform extraction and ethanol precipitation; the intense fast moving band is contaminating RNA: (lane 6 ) pW19 DNA purified using a Qiagen-20 column: (lanes 7 and 8 ) AfnI (lane 7) and Haell (lane 8 ) digested pW19 DNA, same sample as lane 4. The marker is a I-kb DNA ladder from BRL.
resuspended, the bacterial cells are lysed in NaOH/SDS. As described by Birnhiom and Doly,23SDS denatures the cellular proteins, while the alkaline conditions came the chromasomal and plasmid DNA to become denatured. The lysate is neutralized by the addition of acidic potassium acetate. The high salt concentration causes denatured proteins. chromosomal DNA, cellular debris, and SDS to precipitate, while the short plasmid DNA reanneals correctly and stays in solution. The precipitated material is removed by centrifugation, leaving a cleared lysate for binding to the TriplexBeads. The cleared lysate contains plasmid DNA and contaminants including RNAs, unprecipitated chromosomal DNA, polysaccharides, proteins, and other cellular components. The TriplexBeads specifically hind to the plasmid DNA containing the appropriate homopyridinehomopurine insert, allowing its separation and purification. The acidic cell lysate is well-suited to the formation of stable triple-helix DNA. In acidic solution, cytosine is protonated, increasing triple-helix stability (C+.GC).5.2' The lysate is directly mixed with the TriplexBeads. After incubationfor 15min,the beadsarewashedoncewithbinding hufferandoncewiththelow-salt wash bufferwhichstahilizes the triple-helixstructure and minimizes nonspecific binding.= Thelowbufferstrengthofthewash bufferalaohelpstoobtain a high pH in the subsequent elution step. The captureddsDNA iselutedfrom the heads by incubation in a high-pH huffer which disrupts the triple helix without disrupting the douhle-stranded plasmid DNA. The DNA in solution is then ready for immediate use as desired. Characterization of the Purified DNA. A gel electrophoretic analysis of DNAs purified by both conventional methods and the triple-helix capture method is presented in Figure 3. Lanes 1 and 2 are control experiments showing that no DNA is captured if the dsDNA lacka the triple-helixforming sequence (lane 1) or if the capture oligonucleotide is omitted (lane 2). Lanes 3 and 4 show the DNA obtained if the triple-helix complex is formed in solution before addition of heads (lane 3) or, alternatively, if the capture oligonucleotide is immobilized on heads prior to triple-helix formation (lane
I + TriplcxBcads
d-1
ELUTION
Flgun2. SchematlcdlagamoftheMple-heiixmediatedafRlltycaptue
method for dsDNA purification (TAC purification). with a half volume of phenol and a half volume of chloroform. The upper aqueous layer was transferred to a fresh tube and washed twice with an equal volume of chloroform. A half volume of 7.5 M NHIOACand three volumes of ethanol were added and the mixture was cooled at -20 OC for 15 min. The DNA was pelleted by centrifugation, washed twice with ethanol, and redissolved in water.
RESULTS AND DISCUSSION Basic Concept. A diagram outlining this approach to the triple-helix-mediated affinity capture (TAC) purification of plasmid DNA from bacterial cell cultures is shown in Figure 2. The procedure is comprised of three main steps: cell lysis, affinity capture, and elution. After being harvested and
(23) Birnbiom, H. C.; Doly. J. Nucleic Acids Res. 1979, 7,1513-1523. (24) Xodo, L. E.: Manzini, G.: Quadrifoglio, F.: Marel. G. A.; Boom, J. H.Nucleic Acids Res. 1991, 19, 5625-5631. (25) Ji, H.: Smith, L. M., unpublished work. University of Wisconsin-
Madison, 1992.
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ANALYTICAL CHEMISTRY. VOC. 85. NO.
A
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Figure 5. Undigested and Hinull Idigested TACpurlfW plasmid DNAs: (lane 1)pW19DNA;(lanes 2-5)foursubcbnesln pWl9obtalnsd from the Hindlll digest of h phage DNA.
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Flgun 4. Cycle sequencing of T A C - p W dsDNA (A) pW19 DNA sequenced wlthtlw pUC/M13reverseprlmer;(B)pWl9DNAsequenced wkh lha pUC/M13 fwward prlmer; (C) sequence data obtained wkh ilm pUCIM13 forward prlmer from a Haelll subcbne of X in the pW19 vector; (D) conlrol sequence data obtained from pUC19 DNA (0.1 pa). A 5-pL sample of TAG-purlfled DNA was used lor sets A-C: 50 cycles of 95 O C for 10 s and 50 "C lor 30 s were used for the labeling extension wim U - [ ~ ~ P ] ~ A Tfollowed P. by 30 cycles of 95 OC lor 30 s and 72 OC for 2 mln for dMeoxy termination. 4). The quality and yield in both cases appear comparable, indicntingthat the streptavidin-coated beads do not interfere with the formation of the triple-helix structure. Results obtained in two other widely used methods for plasmid purification, phenol-chloroform extraction followed by ethanolprecipitationanduseofanion-exchangecolumn(QiagenZO), are shown in lanes 5 and 6, respectively. Lane 5 is clearly overloaded with a large excess of contaminating RNA, which although generally removed by RNase digestion nonetheless demonstrates the lackofspecificity of thisapproach. Inmany cases, a secondary purification step such as CsCl density
gradient centrifugation is needed to remove such contaminanta. The DNA obtained using the Qiagen column (lane 6) is of generally good quality,26 although RNase digestion is necessary before purification, and small amounts of high molecular weight chromosomal DNA are often observed in these preparations. In contrast, no trace of RNA or chromosomal DNA is observed in the material purified by triplehelix affinity capture (lanes 3 and 4). Lanes 7 and 8 demonstrate the suitability of the triple-helix purified DNA for digestion by the enzymes AjlII and HaeII, suggesting the absence of contaminating species often found to inhibit restriction in impure DNA. Similar results areobtained with avariety of other restriction endonucleases. Taken together, thesedatademonstrate the highdegreeofsequence specificity and low nonspecific binding which characterize the TAC purification method. The high salt concentration (-1.1 M potassium acetate) in the lysis solution may help to minimize nonspecific binding to the magnetic beads.17 The TAC-purified plasmid DNA samples were tested as templates for cycle-sequencing. Good quality autoradiographic data were obtained from the vector pHJ19 using the forward and reverse primers (Figure 4A,B) as well as from a XiHaeIII subclone (Figure 4C). Control sequence data from the plasmid pUCI9 is shown in Figure 4D. Each DNA preparation (1.5 mL of culture and 100 pL of TriplexBeads) was enough for 10 seta of sequencing reactions. Figure 4A also shows the sequence of the inserted triple-helix-forming region. These results show that the TAC-purified DNAs are suitable for direct sequence analysis as eluted from the support. In order to determine whether inserted fragments had any adverse effects upon the TAC purification procedure, subclones were made from a HindIII digest of h phage DNA. Four random subclones were picked and TAC-purified according to the protocol of Table I. Figure 5 shows a gel electrophoretic analysis of the four subclones,both undigested and digested with HindIII to excise the inserted fragments. Three of these four recombinants contained the 6.6kbHindIII fragment from h, whereas the fourth contained a tandem ligation of a 2.0-kb and a 564-bp fragment. All four recombinants werecaptured with similar efficiencyusing the TAC procedure. Another issue of some practical importance concerns the reusuability of the beads. We have found no measurable decrease in capture efficiency in the beads after use over 10 times. The beads appear to be completely regenerated by (26, McCombie. W. R.; Heiner. C.;Kelley. J. M.;Fitzgerald. M. G.; Gocape. J. D.DNA S e q u e n r e J . DNA SPquenrina Mopping 1992,Z. 28S296.
ANALYTICAL CHEMISTRY, VOL. 65. NO.
the elution and washing treatments. This high stability presumably reflects in part the well-documented hardiness of the streptavidin protein.27 The procedure has proven very reliable in our hands. In over 100 preparations of plasmid DNAs with or without inserts, no RNA or chromosomal DNA was ever observed usingfreshorusedTriplexBeads,and every DNApreparation was successfully employed either as a sequencing template or for restriction digestion. About 1h i s needed to prepare theDNAbeginningwith thefreshcellculture, withanaverage yield from a 1.5-mL culture of - 2 fig of DNA. The capture efficiencies of supercoiled circular dsDNA (uncut pHJ13) and linearized dsDNA (HindIII-digested pHJ13) were measured. Over 80% of the supercoiled dsDNA was captured using the purification steps in Table I as estimated by gel electrophoresis, while a very low yield (less than 20% ) was obtained for linearized dsDNA. This result differs from that reported previously,'7 in which linearized DNAs were captured with slightly higher efficiencies than were supercoiled DNAs. Although the reason for this difference is not known, it may relate to the details of the particular triple-helix sequence and buffer conditions employed. The supercoiling may change the conformation of the DNA, possibly exposing the triple-helix region or, alternatively, reducing the size of the molecule in solution and hence minimizing steric interactions with the support particle. If the triple-helix structure is formed in solution prior to binding to beads, both linear and supercoiled DNAs are captured with similar efficiencies of -80%. Optimization of t h e Purification. This approach to DNA purification is based on two distinct binding processes. Thefirstistheformationofatriple-helicalstructure,aprocess which has been extensivelystudied,24,2~30with respectto both the kinetics and the thermodynamics of the process. Triple helices have been formed with DNAs of widely varying size, ranging from small plasmids5 to megabase chromosomes? and have found a wide variety of applications. The second aspect is the capture of the triple-helical complex. This was achieved here by means of the high-affinity interaction of biotin to streptavidin ( K , = 10-15 M Y immobilized on magnetic beads. We foundthat, in contrastto previous work, in which the two steps of triple-helix formation and solidphase immobilization were performed separately overa period of 3 h,'7 the two procedures can be combined into a single step requiring only 15 min. An additional 10 min is needed to elute the DNA, giving a duration for the entire process of under 30 min. Several parameters of the procedure were examined for the purpose of optimization. The amounts of both beads and oligonucleotide were varied to maximize recovery. The manufacturer's specifications indicated that 1mg (100 fiL) of beads could capture between 30 and 300 pmol of biotinylated DNA, depending upon the fragment size.3' This is equivalent to 40 fig of a 2-kb DNA. Figure 6 shows a gel electrophoretic analysis of the results obtained by varying the amount of beads (lanes 1-4) or the amount of capture oligonucleotide (lanes 7-10). In these experiments, either the amount of oligonucleotide was held constant while the amount of atreptavidin beads was varied or, conversely, the amount of beads was held constant while the amount of (271 Wilchek, M.; Bayer. E. A. Auidin-Biotin Technology; Academic Press: San Diego, CA, 1990. (281 Pi1eh.D. S.;Brousseau,R.:Shafer,R.H.Nucleic Acids Res. 1990, 18,5743-5150. (29)Plum, G.E.;Park, Y.-W.; Singleton. S.F.; Dervan, P. B. h o e . Nafl. Aead. Sei. U.S.A. 1930.87, 9436-9440. (30)Maher. L. J.; Dervan, P. B.; Wold, B. J. Biochemistry 1390,29, 882WRRZR. (311 Dynalbeods M-280TeehniealHnndbook:Dynal,Ioe.:Great Neck, ~~~~
~~~~
NY. 1989.
Eeads(plI)
20 50100200
Probe (pnd)
20 20 20 20
IO. MAY 15, 1993 1327 100100100100 0 5 20 50
Flgura 6. pWl9 DNA captured with varying amounts of beads and capture ollgonucleotlde: (lanes 1-4) 20-200 FL of beads, 20 p m l oligonucleotide: (lanes 5 and 6 ) and 0.2 and 0.4 pg of pKll9 DNA, respectively; (lanes 7-10) 100 pL of beads, 0-50 pmol of capture oli(1onuc1eotMe. The DNA was eluted in 20 ILL of elution buffer. from w6ch 2 pL was loaded into the gel
oligonucleotide probe was varied. In both cases, the capture oligonucleotide was bound to the beads prior to their use and unbound material removed prior to use by washing as described above. The amount of beads employed had a strong effect upon the results. More DNA was obtained as more beads were employed, in spite of the fact that the nominal capacity of thebeads wasmuchgreaterthan theamountofDNAactudy bound. Although this would suggest that the effectof adding more beads was kinetic in nature, experiments in which more time was allowed for binding to the beads did not result in a higher yield. Given this result, it seems likely that the capacity of the beads for the plasmid DNAs is much less than the manufacturers's reported capacity, presumably due to the relatively large size. As increasing the amount of beads used increases the cost of the procedure and also demands that a larger volume of elution buffer be employed, diluting the final DNA solution, a compromise of 100fiL of beads was chosen for routine use (Table I). Incontrasttothesensitivityoftheprocedure totheamount of beads employed, the amount of capture oligonucleotide employed had little effect. This suggests that the binding is limited by the number of sites on the beads accessible to the supercoiledtarget molecule, which is probablyonly a fraction of those accessible to the much smaller biotinylated capture oligonucleotide. The above results suggested that the accessibility of the hiotin group and capture oligonucleotide were likely to be important factors in the system. Accordingly, the length of the polynucleotide stretch between the biotin group and the homopyridine stretch was varied. Three 20-base homopyridine probes with spacers of 0,5, and 10 bases were tested. The 5-base and 10-base spacers both yielded similar capture efficiency, while the prohewithout aspacergave amuchlower efficiency (data not shown). Another factor of possible significance is the length of the triple-helical region employed for capture. The stability of the triple-helix region increases with length,5suggesting that a higher yield might be obtained with a longer capture sequence. Three probes with triple-helix stretches of 15,20, and 32 bases were tested (see the Experimental Section for the exact structures). It was found that the 20-base oligonucleotide gave the highest captureefficiencyfor pHJ13DNA (data not shown). Although the lower efficiency of the 15base stretch is easily rationalized on the basis of its lower stability, the reason for the lower efficiency of the 30-base probe is less clear.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
The last step of the TAC purification is elution of the DNA from the beads. Triple-helix DNA is unstable at high pH because of the deprotonation of cytosine32and may thus be eluted from the beads by adjusting to pH 9.0.” In order to maximize the versatility of the eluted DNA solution, rendering a desalting step unnecessary, the concentration of the elution buffer was kept as low as possible. Buffers of Tris-HC1, pH 9.0, at concentrations of 1000, 100, 50, 20, and 10 mM were tested for DNA elution. Little difference was observed for concentrations from 1000 to 50 mM, whereas a substantially lower elution efficiency was observed for 20 and 10 mM buffers. Similar behavior was observed at pH 8 and 10. An elution buffer of 50 mM Tris-HC1, pH 9.0, was accordingly chosen. This elution buffer was found to be suitable for direct enzymatic digestion and DNA sequencing as shown above. TAC Purification of X Phage DNA. In order to test the applicability of this triple-helix-mediated affinity capture to larger DNAs, a triple-helix capture sequence was inserted (32) Callahan, D. E.; Trapane, T. L.; Miller. P. S.;Ts’o, P. 0. P.; Kan. L.-S. Biochemistn 1991, 30, 165CF1655.
into XgtlO DNA (46 kb) as described above to give the recombinant X derivative XgtlOT. For these larger DNAs, higher binding efficiency is obtained when the triple helix is formed in solution prior to capture on the streptavidin beads. This is similar to the behavior of the linearized plasmid DNA described above. Under these conditions, a -60% yield of XgtlOT DNA was obtained for purified X phage DNA. A yield comparable to that obtained using an widely used ionexchange procedure (see Materials) was obtainedfrom XgtlOT phage lysates. The optimization of this procedure and its possible extension to larger DNAs such as YAC chromosomes are under investigation.
ACKNOWLEDGMENT This work was supported by DOE Human Genome Initiative Grant DE-FG02-90ER61026. We thank Dr. David Mead and Dr. Richard Guilfoyle for many helpful discussions, and Dr. Guifoyle for providing the X DNAiHaeIII fragments.
RECEIVED for review October 8, 1992. Accepted January 4, 1993.
