Combining the Preparation of Oligonucleotide Arrays and Synthesis … · 2000-11-24 ·...

ANALYTICAL BIOCHEMISTRY 243, 218–227 (1996)ARTICLE NO. 0509

Combining the Preparation of Oligonucleotide Arrays andSynthesis of High-Quality Primers

Jan Weiler1 and Jorg D. HoheiselMolecular–Genetic Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506,D-69120 Heidelberg, Germany

Received June 27, 1996

purposes. For clinical applications like the antisenseBased on the oligomer-chip technology, oligonucleo- strategy (reviewed in Ref. 2), gram or even kilogram

tide arrays were synthesized directly on polypropyl- amounts of oligonucleotides are required, promptingene sheets by a modified phosphoramidite chemistry efforts on an up-scaling of synthesis yields (e.g., 3). Forusing b-eliminating nucleobase-protecting groups in many applications in molecular biology, on the othercombination with a succinate solid-phase linker. This hand, notably DNA characterization and screening bymethod decouples the oligonucleotide deprotection hybridization and enzymatic assays like the polymer-from the support cleavage procedure, in contrast to ase chain reaction (PCR) and DNA sequencing, oligo-standard phosphoramidite chemistry. In addition to mer quantities in the picomole range are usually anbeing reliable substrates for hybridization experi- adequate amount, but larger numbers of oligonucleo-ments, the arrays also serve as source for the isolation tides are needed. Again, this has led to procedures aim-of individual oligonucleotides. Technically, this al- ing at the simultaneous production of different oligonu-lowed for a direct control of the quality of the arrayed

cleotides in small quantities (e.g., 4, 5).oligomers. The released compounds were sufficient inMore recently, another consequential technology inamount and purity to work without further purifica-

molecular biology was developed using ordered arraystion in PCR and DNA-sequencing reactions, with theof oligonucleotides to serve as analytic and diagnosticresults being identical to controls with commerciallytools for DNA analyses (6–8). This oligomer-chip tech-obtained primer molecules. Consequences for oligo-nology has a huge potential to play an important rolemer-chip hybridization procedures, the applicabilityin many aspects of genome analysis (for a review seeof such hybrid-function arrays in, for example, diag-Ref. 9). Technically most challenging is the sequencingnostics or comparative biology, and developments to-by hybridization (SBH)2 strategy, whereby the se-ward parallel primer synthesis are discussed. q 1996

Academic Press, Inc. quence of an unknown nucleic acid is reconstructedfrom its hybridization binding pattern on a matrixwhich contains a comprehensive set of short oligonucle-otide sequences (e.g., all 65,536 octamers).

Initiated by the combination of solid-phase technol- During our work concerned with developments to-ogy and the phosphoramidite chemistry introduced by ward a practical application of the SBH technique, suchBeaucage and Caruthers (1), there has been much prog- as the ordering and selection of DNA fragments suit-ress in the automation of DNA synthesis. Today, the able for SBH analysis (10, 11), for example, or the se-preparation of synthetic oligonucleotides needed in bio- quence-independent leveling of oligomer binding sta-logical, biomedical, and physical applications is usually bilities (12), it was apparent to us that such oligomerexecuted with commercial, automated DNA synthe- chips by design would be an ideal tool not only forsizers. These machines produce oligonucleotides in the screening procedures but also for the synthesis ofnanomole up to micromole range. However, over thepast few years, there have been two diametrical tend- 2 Abbreviations used: SBH, sequencing by hybridization; TOTU,

O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N*,N*-tetramethyl-encies with regard to oligomer synthesis for biomedicaluronium tetrafluoroborate; DBU, 1,8-diazabicyclo-(5.4.0)-undec-7-ene; NPE, 2-nitrophenylethyl; NPEOC, 2-(4-nitrophenyl)ethoxycar-bonyl; POM, polyoxymethylene; DMF, dimethylformamide; THF,1 To whom correspondence should be addressed. Fax: /49-6221-

424682. E-mail: [email protected]. tetrahydrofuran.

218 0003-2697/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

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COMBINING OLIGOMER ARRAYS AND PRIMER SYNTHESIS 219

primer molecules, if quantity and purity of each indi-vidual oligomer would be sufficient for biological reac-tions. For this purpose, a modification of the synthesischemistry was needed that would allow the use of theoligomer grids both for hybridization experiments andfor the isolation of the individual oligomers. To thisend, the deprotection step after completed oligonucleo-tide synthesis had to be disconnected from the step ofcleaving the oligonucleotides off the support medium,with both manipulations taking place at high effi-ciency. Here we describe the establishment of such asynthesis procedure and demonstrate the suitability ofthe released oligonucleotides in amount and purity forPCR amplification and DNA sequencing.

MATERIALS AND METHODS

Reagents. O-[(Ethoxycarbonyl)cyanomethylen-amino]-N,N,N*,N*-tetramethyluronium tetrafluoroborate(TOTU), 1,6-bis-(methylamino)-hexane, p-nitrophenyl-chloroformate, N-methylmorpholine, 1,8-diazabicyclo-(5.4.0)-undec-7-ene (DBU), N-methylimidazole, t-butylhydroperoxide, and solvents were purchased from Flukaand used without additional purification. Amino-function-alized polypropylene sheets were obtained from Beckman;5*-O-DMT-2*-deoxynucleoside-3*-O-(b-cyanoethyl)-N,N-diisopropyl-phosphoramidites protected by 2-nitrophenyl-ethyl(NPE) and 2-(4-nitrophenyl)ethoxycarbonyl(NPEOC) functions was from Chemogen (Constance, Ger-many). If not indicated otherwise, oligonucleotides used ashybridization probes or as controls in enzymatic reactions

FIG. 1. Drawing of the reaction chamber used for oligonucleotidewere purchased from MWG Biotech (Ebersberg, Ger-synthesis on polypropylene sheets. The device is actually square inmany). [g-32P]ATP (10 mCi/ml), T4 polynucleotide kinase,format to allow 907 rotations of the polypropylene sheet betweenand buffers for labeling oligomers at their 5*-end were synthesis steps.

from Amersham and New England Biolabs, respectively.Chamber for the synthesis of oligonucleotide arrays.

For the initial experiments, a simple polyoxymethy- ture of pyridine and acetic anhydride for 2 h, cappinglene (POM) block was constructed containing eight amino groups remaining on the surface. Subsequently,channels of 1 mm depth, 70 mm length, and 4 mm the sheets were washed with dichloromethane andwidth each, with two holes at both ends for connection taken up in 50 ml acetonitrile or dimethylformamideto the inlet and outlet of a commercial DNA synthe- (DMF); 0.2 ml 1,6-bis-(methylamino)-hexane wassizer. The polypropylene film was held in place by a added and the mixture was shaken at 407C for 48 h.silicon seal and a lid of perspex screwed to the POM After washing successively with DMF, methanol, andbasis (Fig. 1). For perfect sealing, additional pressure acetone, the methylamino-modified polypropylene waswas applied to the whole apparatus by a clamp. By dried and stored at 47C.removing the polypropylene sheet after a single or sev- Derivatization of methylamino-modified support witheral cycles, rotation by 907, and continued synthesis, nucleosides. 3*-O-Succinate derivatives of protectedthis simple and small instrument allowed the synthe- nucleosides (dANPEOC, dCNPEOC, dGNPEOC/NPE, dT, andsis of up to 64 oligomers. fluorescein-labeled dC [Å dCfl; Ref. 13]) were prepared

by the method of Kierzek et al. (14) and were loadedPreparation of methylamino-modified polypropylenesupport. In a typical experiment, aminated polypro- to the methylamino-modified polypropylene after

mounting a sheet in the synthesis chamber. Five milli-pylene sheets (8 1 8 cm2) were gently shaken in a mix-ture of 25 ml dry dichloromethane, 25 ml dry dioxane, grams of the desired nucleoside-3 *-succinate was mixed

with 25 ml N-methylmorpholine and 10 ml acetonitrile,0.2 g p-nitrophenylchloroformate, and 160 ml triethyla-mine for 2 h at room temperature. After washing with before adding 4 mg of TOTU. The bottle with the mix-

ture was immediately connected to the DNA synthe-dichloromethane, the sheets were shaken in a 1:1 mix-


WEILER AND HOHEISEL220

FIG. 2. Probe attachment chemistries and deprotection by ammonia (NH4OH) used for standard synthesis of free oligonucleotide (a) andthe preparation of oligonucleotide arrays (b); X Å O (16); X Å NH (17).

sizer and a simple preprogrammed cycle was activated: about 1 Mcpm radioactively labeled oligomer probe(concentrationÅ 6 pmol/ml) for 18 h at 47C. After wash-first the rows were rinsed with the reaction mixture

and the reaction was kept for 30 min; then, the re- ing for 30 min at 47C, autoradiography was at 0707Cusing intensifying screens. Probes were stripped off theagents were flushed out with argon and the rows were

washed with acetonitrile. To block residual meth- sheets by incubation in hybridization buffer at 657Cfor 3 h.ylamino groups on the polypropylene, capping solution

as described below was applied. Alternatively, the de- Releasing oligonucleotides from the polypropylenerivatized polypropylene membranes were removed support. For cleaving off individual oligonucleotides,from the chamber and shaken in a mixture of 10 ml the desired square of an oligomer array on a polypropyl-acetic anhydride, 10 ml dry pyridine, and 1 ml N-meth- ene sheet was cut out with a key punch machine. Afterylimidazole for 2 h in a polypropylene box. After succes- incubation in 30% aqueous ammonia for 2 h, the re-sive washing with DMF, methanol, and acetone, the leased oligonucleotide product was lyophilized andsheets were dried and stored at 47C until usage. used for PCR and DNA sequencing without further pu-

Oligonucleotide synthesis. Oligonucleotide synthe- rification.sis on polypropylene sheets was carried out as detailedin Table 1. After completed synthesis, the sheet was

RESULTSremoved from the chamber and shaken in 1 M DBU inacetonitrile in a polypropylene box at 407C overnight Usually, for oligonucleotide synthesis the commonfor oligomer deprotection. The membrane was washed b-cyanoethyl phosphoramidite chemistry is used: thewith acetonitrile and acetone, before being used for hy- phosphorus of the monomer building blocks is pro-bridization experiments or cleavage of the oligomers, tected with a b-cyanoethyl function, and acyl function-respectively. No deterioration of experimental results alities like benzoyl-(dAbz, dCbz), acetyl-(dCac), or isobu-was observed even after storing the oligomer arrays tyryl-groups (dGibu) are used for nucleobase blocking.over several weeks at room temperature. After completion of the oligonucleotide chain elonga-

tion process, the protecting groups are removed nucleo-Hybridization with oligonucleotide probes. Oligonu-cleotide probes were end-labeled with [g-32P]ATP un- philically by incubation in concentrated aqueous am-

monia at 607C (Fig. 2a). The difference between theder standard conditions (15). The oligomer arrays wereprehybridized in 600 mM NaCl, 60 mM sodium citrate, synthesis of free oligonucleotides in commercial ma-

chines and the construction of oligomer arrays (16, 17)pH 7.5, 7.2% sodium N-lauroylsarcosine for 1 h andthen incubated in 10 ml of the same solution containing is the anchoring to the solid support at the 3*-terminal



FIG. 3. Separation of nucleobase deprotection of solid-phase-bound oligonucleotides from support cleavage. Application of the NPE/NPEOCstrategy allows alternative use of the oligomer arrays for either hybridization experiments (option I) or as a source for the isolation ofindividual primer molecules (option II).

nucleoside. While for the former an ester bridge like ments with the capability of cleaving the oligonucleo-tides off the support, to be used as probes or primerthe succinate linker is intentionally cleaved simultane-

ous to the biopolymer deprotection, the arrayed oligo- molecules for enzymatic reactions like PCR or DNAsequencing, for example, nucleobase deprotection hadnucleotides must remain attached to the solid phase

after the deprotection process for hybridization experi- to be decoupled from the release reaction (18), withnevertheless stable binding during oligomer synthesisments. In earlier experiments, this was achieved by

linkage via a stable phosphoester (16) or phosphorami- and any subsequent hybridization experiment. Thisgoal was reached by using the common succinate linkerdate (17) bond (Fig. 2b).

For the preparation of oligonucleotide hybrid arrays between the 3*-terminal nucleoside and the solid phasein combination with an alternative phosphoramiditethat combine usage in hybridization screening experi-



FIG. 4. Activation of the methylamino-modified polypropylene sheets and coupling of appropriately protected nucleosides.

approach, the so-called ‘‘NPE/NPEOC strategy’’ (19), In Situ Oligonucleotide Synthesiswhich on CPG and polystyrene supports had led to oli- Initially oligomer synthesis on the polypropylenegonucleotide products of high purity (20, 21). This syn- sheets with the NPE/NPEOC nucleoside derivativesthesis strategy uses the b-eliminating base-protecting was carried out under running standard phosphora-groups NPE and NPEOC. These functions allow the midite chemistry protocols. In subsequent PCR, how-deprotection of the biopolymer by the strong but nonnu- ever, such primers could not equal the performancecleophilic base DBU while the oligonucleotide remains obtained with primer molecules of identical sequenceattached to the polypropylene (Fig. 3). that were synthesized as a control on CPG material

using the same chemistry. Intensive studies of the syn-Surface Modification thetic conditions indicated that mainly the tetrahydro-

The chemistry used for synthesis required an anchor- furan (THF) solvent included in standard capping anding structure between the oligonucleotides and the oxidation solutions was responsible for low couplingaminated polypropylene sheets which is cleavable un- yields and/or product contamination on the polypropyl-der nucleophilic conditions, i.e., by treatment with con- ene support. Most probably, a swelling of the polypro-centrated aqueous ammonia, but remains stable under pylene film in the presence of THF (R. Matson, personalthe nonaqueous DBU deprotection conditions. In accor- communication) obstructed the complete removal of un-dance with results reported earlier (22), we found it reacted reagents, thus leading to largely decreased cou-necessary to introduce an alkylamino-modified spacer pling yields. Much better results were achieved whenbetween the primary amino group of the polypropylenesheet and the succinate linkage to meet these require-ments (Fig. 4). The yield of this activation step wasdetermined by p-nitrophenolate liberation (23) to rangebetween 30 and 90 pmol/cm2.

TABLE 1

Protocol Used for the Production of Oligonucleotide Arrayson Polypropylene Membranes Using the Eight-Channel Syn-thesis Chamber Connected to an ABI-392 DNA Synthesizer

Step Reagents or solvent Time (s)

Washing Acetonitrile 30Detritylation 3% TCA in dichloromethane 80Washing Acetonitrile 210Coupling 75 mM phosphoramidite / 40

0.5 M tetrazole inacetonitrile

Washing Acetonitrile 30Capping (I / II) Ac2O/N-methylimidazole in 50

acetonitrileOxidation 1 M t-butyl hydroperoxide in 130

acetonitrile FIG. 5. Capillary electropherogram of a 15-mer oligonucleotideWashing Acetonitrile 120 that had been synthesized on a polypropylene sheet. Electrophoresis

was carried out in a gel-filled tube, and detection was via a fluoresceinTotal 690label at the 3 *-terminal base.



FIG. 6. Typical hybridizations to oligomer arrays on polypropylene membranes. (a, b) Four different starting nucleosides, dCNPEOC (columns3 and 7), dANPEOC (columns 2 and 6), dCbz (column 1), and dCfl (columns 4 and 8), were coupled to a polypropylene sheet using the eight-channel synthesis device. The sheet was removed from the chamber and rotated 907. Subsequently, seven different oligonucleotides weresynthesized (A–H) creating 49 squares containing 28 different sequences. One channel was left empty in both dimensions as a control(lanes 5 and D). After oligomer deprotection, some squares (e.g., B2, B6, B8, C8, and E8) were cut out for analyses. The remaining sheetwas hybridized with (a) the 9-mer d(CTATAGTGA) and (b) the 12-mer d(GA11). Complementary sequences are underlined. In (c) and (d),the sheet had been turned 907 in between synthesis steps. Nonamers whose sequences cover breakpoints of synthesis were hybridized.

the standard capping reagents (capping I: THF/2,6-lut- in acetonitrile was applied for oxidation. The final pro-tocol used for oligonucleotide synthesis on polypropyl-idine/Ac2O, 8:1:1; capping II: N-methylimidazole/THF,

16:84) were replaced by a 1:1 mixture of Ac2O and ace- ene is shown in Table 1. Approximately 300 ml of 75mM phosphoramidite solutions was applied for eachtonitrile (capping I) and a 2:8 mixture of N-methylimid-

azole and acetonitrile (capping II). Furthermore, in- coupling step so that the coupling reagents were ingreat excess over the growing nucleotide chain on thestead of iodine, a 1 M solution of t-butyl hydroperoxide



Hybridization Experiments

First, to confirm the stability of the succinate linkerduring the DBU deprotection and hybridization condi-tions and, second, to test the selectivity of hybridiza-tions to the polypropylene matrices, oligomer arrayswere used for hybridization experiments, in some casesafter individual oligomers had been removed for otheranalyses. Each sheet was briefly washed in hybridiza-tion buffer prior to hybridization experiments with ra-dioactive probes. Duplex formation was detected by au-toradiography. Typical results on test grids arepresented in Fig. 6. The succinate linkage was foundto be stable during DBU deprotection and hybridiza-FIG. 7. PCR amplification of a plasmid insert. Both commercialtion and stripping procedures. Arrays were used in fiveprimers were used for the reaction run in lane A. In lanes B and C,subsequent hybridization experiments without appar-either of the primer molecules had been substituted for by oligonucle-

otide isolated from 0.16 cm2 of a polypropylene sheet. Marker (lane ent loss of signal. Oligomer probes were applied thatD) is HindIII-digested l-DNA and plasmid pUC18 cut with FspI. were complementary to either the 5*-end (Fig. 6a) or

the 3*-end (Fig. 6b) of individual oligonucleotides onthe grid. Positive signals by the latter proved the acces-polypropylene surface. This new kit of reagents for sibility to hybridization of the most 3*-position of aDNA synthesis also succeeded in producing high-qual- surface-bound oligonucleotide, the former confirmedity oligonucleotides in common solid-phase syntheses the presence of the 5*-end. The equivalent signal inten-on CPG support in the 0.2-mmol scale. Examination of sities detected with both probes suggested high cou-the products by analyses based on HPLC separation pling yields during synthesis, thus confirming the re-and capillary electrophoresis did not find any differ- sults of the more stringent quality checks on releasedences in quality—such as the occurrence of base modi- oligomers. One base pair mismatch selectivity wasfications—compared to standard synthesis protocols. achieved by hybridizing to target sequences as shortas hexamers, even if there was sequence repetition andOligomer Qualitythe difference between the corresponding terminal nu-

Oligonucleotides synthesized on polypropylene were cleotide was only a substitution to the nucleobasedeprotected by a DBU treatment and subsequently re- (Fig. 6b).moved by ammonia from the surface of the support foranalyses of purity and quantity. The appropriate piece

PCR and Sequencing Applications of Releasedof a polypropylene film was cut out, and the oligomerOligonucleotideswas released by a treatment in 30% aqueous ammonia.

Lyophilized, the DNA was taken up in water. Oligonu- For enzymatic reactions, oligonucleotide primerswere removed from the polypropylene and taken up incleotides of up to 29 bp in length were labeled either

at the beginning of an in situ synthesis at the eventual water without further purification. PCR on recombi-nant plasmid pTZ18R was done under standard condi-3 *-terminal base by incorporation of a fluorescein-la-

beled cytosine (13) as the starting nucleoside or by tions as described in detail earlier (11). Primers were26- and 29-mers binding to the vector directly adjacenttransfer of a radioactive phosphate group to the 5*-end

of a released oligonucleotide using T4 polynucleotide to either side of the insert DNA. An oligonucleotideamount equivalent to an area of 0.16 cm2 of the polypro-kinase. Analysis of molecule length was by direct fluo-

rescence detection after capillary electrophoresis in a pylene surface was used in reactions of 25 ml. Two-temperature PCR was done, primer annealing and ex-Beckman P/ACE 5000 machine (e.g., Fig. 5) or autora-

diography subsequent to polyacrylamide gel electro- tension at 687C and strand denaturation at 957C. Onagarose gels, the products were compared to the resultsphoresis (not shown), respectively. With the final, mod-

ified synthesis protocol (Table 1), most of the obtained with commercial primer molecules used at aconcentration of 1 mM. No significant difference in qual-oligonucleotide product was found to be full-length mol-

ecules indicating a stepwise coupling efficiency of more ity or quantity of the PCR products could be observed(e.g., Fig. 7).than 98.5%. From an evaluation of the signal intensi-

ties detected in the same experiments, the amount of The ultimate test of the coupling efficiency duringsynthesis, and hence oligonucleotide quality, is its usereleased oligonucleotide probe could be calculated to be

more than 25 pmol/cm2 polypropylene. This value is in as primer in an enzymatic sequencing reaction. Evenrelatively small contaminations by shorter side prod-good agreement with the 30- to 90-pmol range deter-

mined for the initial activation step prior to synthesis. ucts would lead to extra bands in the sequencing gel.



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AID AB 9818 / 6m22$$9818 11-12-96 16:11:13 aba AP: Anal Bio


In a typical experiment, the 18-mer ‘‘forward’’ primer a detector molecule, immediately followed by its usefor an isolation of the appropriate DNA fragment byd(GTAAAACGACGGCCAGTG) of the bluescript plas-

mid was isolated from polypropylene and used in se- PCR or a direct tag sequence analysis of the region.Also, oligonucleotides identified by hybridization of aquencing reactions. Oligomer taken from 0.32 cm2 was

used per standard cycle sequencing reaction. The qual- given DNA could in turn be used as probes for theidentification of homologous fragments, for example,ity of the sequencing profiles was compared to reactions

using commercial primer. Again, no apparent differ- an approach that would simplify and speed up compar-ative genome analyses. For some applications, it mightence in performance could be detected (Fig. 8).be necessary to introduce a stretch of unspecific basesat the 3* terminus in order to ensure an enzymatic

DISCUSSION extension of the molecules by a polymerase. Due tocombinatorial constraints, only a moderate number ofA modified phosphoramidite chemistry on a polypro-

pylene membrane as solid support was established that sequence variations can be synthesized in parallel ona device of a type as depicted in Fig. 1. Nevertheless,not only allows oligonucleotide arrays to be used as

substrates in hybridization experiments but also offers such kind of array is of interest for diagnostic applica-tions, for example. Moreover, the results suggest thatthe opportunity to separate each compound from the

synthesized oligomer grid. With regard to the SBH ultimately even a larger number of independent oligo-nucleotides could be generated by an appropriately ad-technology, this provides the means for a direct quality

assessment of the oligomers synthesized on the array, justed device. This could make techniques that requiresmall amounts of different oligonucleotides such asrather than to use results of hybridization experiments

for such evaluation. Recent investigations (24) showed primer walking sequencing even more functional.that the reliability of hybridizations to arrays of shortoligonucleotides depends strongly on the quality of the ACKNOWLEDGMENTSarray-bound oligomers. Hence, both the high-quality

We thank Sandra Schwarz for excellent technical assistance, Ste-oligonucleotides on the chip and the independent con-phen Case-Green and Edwin Southern for help in establishing thetrol mechanism provided by the presented technique technique of oligonucleotide array construction and useful discus-

are paramount for accurate SBH analysis. sions, Robert Matson of Beckman Instruments for providing ami-nated polypropylene sheets and related information, WernerSome further improvements concerning the chemis-Fleischer and Wolfgang Schutz for generously letting us work withtry are currently being investigated: preliminary ex-their DNA-synthesizer, and Achim Karger for analyses by capillaryperiments indicate, for example, that the time of oligo- electrophoresis. The work was funded by the Deutsche Forschungs-

mer production could be shortened drastically, in gemeinschaft.particular by abbreviating the period of the DBU depro-tection process from 12 to 1 h at higher reaction tem- REFERENCESperatures. Also, technical aspects require further de-

1. Beaucage, S. L., and Caruthers, M. H. (1980) Tetrahedron Lett.velopment. The eight-channel device had only been22, 1859–1862.designed as a very simple tool for initial analyses. How-

2. Crooke, S. T. (1995) Hematol. Pathol. 9, 59–72.ever, key issues could be tested, such as the flow of3. Wright, P., Lloyd, D., Rapp, W., and Andrus, A. (1993) Tetrahe-chemicals through the channels or the sealing mecha- dron Lett. 34, 3773–3776.

nism. From this basis, construction of much more re- 4. Sindelar, L. E., and Jaklevic, J. M. (1995) Nucleic Acids Res. 23,fined instruments for different applications is under- 982–987.way. Another problem that is being addressed is the 5. Lashkari, D. A., Hunicke-Smith, S. P., Norgren, R., Davis, R. W.,control of the synthesis cycles. Although a commercial and Brennan, T. (1995) Proc. Natl. Acad. Sci. USA 92, 7912–

7915.machine was sufficient on a small scale, modified in-6. Khrapko, K., Lysov, Y., Khorlyn, A., Shick, V., Florentiev, V.,struments are needed for automatically serving larger

and Mirzabekov, A. (1989) FEBS Lett. 256, 118–122.arrays. Concomitant, technical advances are in prog-7. Southern, E. M., Maskos, U., and Elder, J. K. (1992) Genomicsress to reduce substantially the consumption of re-

13, 1008–1017.agents by adapting the tubing and vent system toward8. Caviani-Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M. J.,a reagent delivery system that is more effective than

Holmes, C. P., and Fodor, S. P. A. (1994) Proc. Natl. Acad. Sci.a standard DNA synthesizer. USA 91, 5022–5026.

The complete and efficient separation of nucleobase 9. Mirzabekov, A. D. (1994) Trends Biotechnol. 12, 27 –32.deprotection and release from the solid support, the 10. Hoheisel, J. D. (1994) Trends Genet. 10, 79–83.stability of the succinate linker during hybridization, 11. Scholler, P., Karger, A. E., Meier-Ewert, S., Lehrach, H., Delius,and the high quality of the oligomer molecules suggest H., and Hoheisel, J. D. (1995) Nucleic Acids Res. 23, 3842–3849.a combined use of the arrays both as an instrument for 12. Hoheisel, J. D. (1996) Nucleic Acids Res. 24, 430–432.screening experiments and as source for oligonucleo- 13. Maier, T., and Pfleiderer, W. (1993) Collect. Czech. Chem. Com-

mun. 58, 209–213.tide primers. Thereby, an oligomer could first serve as



14. Kierzek, R., Caruthers, M. H., Longfellow, C. E., Swinton, D., 19. Stengele, K. P., and Pfleiderer, W. (1990) Tetrahedron Lett. 31,2549–2552.Turner, D. H., and Freier, S. M. (1986) Biochemistry 25, 7840–

7846. 20. Weiler, J., and Pfleiderer, W. (1995) Nucleosides Nucleotides 14,921–924.

15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular21. Weiler, J., and Pfleiderer, W. (1996) in Innovations and Perspec-Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory

tives in Solid Phase Synthesis and Combinatorial Chemical Li-Press, Cold Spring Harbor, NY.braries (Epton, R., Ed.), Mayflower Worldwide, Edinburgh, inpress.16. Maskos, U., and Southern, E. (1992) Nucleic Acids Res. 20, 1679–

1684. 22. Brown, T., Pritchard, C. E., Turner, G., and Salisbury, S. A.(1989) J. Chem. Soc. Commun., 891–893.

17. Matson, R. S., Rampal, J., Pentoney, S. L., Anderson, P. D., and23. Katzhendler, J., Cohen, S., Rahamim, E., Weisz, M., Ringel, I.,Coassin, P. (1995) Anal. Biochem. 224, 110–116. and Deutsch, J. (1989) Tetrahedron 45, 2777–2792.

18. Albretsen, C., Kalland, K.-H., Haukanes, B.-I., Havarstein, 24. Pirrung, M. C., and Bradley, J.-C. (1995) J. Org. Chem. 60, 6270–6276.L.-S., and Kleppe, K. (1990) Anal. Biochem. 189, 40–50.


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