Proc. Natl. Acad. Sci. USAVol. 91, pp. 5022-5026, May 1994Biochemistry

Light-generated oligonucleotide arrays for rapid DNAsequence analysis

(sequendng by hybrdhzaton/comblnatorlal chemistry/DNA diocs)

ANN CAVIANI PEASEt, DENNIS SOLASt, EDWARD J. SULLIVANt, MAUREEN T. CRONIN*,CHRISTOPHER P. HOLMESt, AND STEPHEN P. A. FODORt#*Affymetrix, 3380 Central Expressway, Santa Clara, CA 95051; and tAffymax, 4001 Miranda Avenue, Palo Alto, CA 94304

Communicated by Ronald W. Davis, January 4, 1994

ABSTRACT In many areas of molecular biology there is aneed to rapidly extract and analyze genetic information; however,current technologie for DNA sequence analysis are slow andlabor Intensive. We report here how modern photlithographictechniques can be used to facilitate sequence analysis by gener-ating miniatuiz arrays of densely packed olgonuceotideprobes. These probe arrays, or DNA chips, can then be appliedto p i DNAhybznanalysis, directiy yielding sequenceinformaton. In ap i experiment, a 1.28 x 1.28cm arrayof 256 dfferent o tides was produced in 16 chemicalr cycles, ruiring 4 hr to complete. The hybridizationpatternOf fuorescendy labeled olignuleotide targets was thendetected by epffluorescence miscopy. The fluorescence signaisfrom c mary probes were 5-35 times stronger than thosewith single or double base-pair hybridiztn misatches, dem-onstrating s it in the identication of comple try se-quences. This Method should prove to be a powerfMl tool for rapidnv s in human genetics and d nostics, pathogen de-tection, and DNA mdecular recognition.

Conventional DNA sequencing technology is a laborious pro-cedure requiring electrophoretic size separation of labeledDNA fragments. An alternative approach to de novo DNAsequencing, termed sequencing by hybridization (SBH), hasbeen proposed (1-3). This method uses a set of short oligonu-cleotide probes of defined sequence to search for complemen-tary sequences on a longer target strand ofDNA. The hybrid-ization pattern is then used to reconstruct the target DNAsequence. It is envisioned that hybridization analysis of largenumbers of probes can be used to sequence long stretches ofDNA. In more immediate applications of hybridization meth-odology, a small number of probes can be used to interrogatelocal DNA structure.The strategy of SBH can be illustrated by the following

example. A 12-mer target DNA sequence, AGCCTAGCT-GAA, is mixed with a complete set of octanucleotide probes.If only perfect hybridization complementarity is considered,5 of the 65,536 octamer probes-TCGGATCG, CGGAT-CGA, GGATCGAC, GATCGACT, and ATCGACTT-willhybridize to the target. Alignment of the overlapping se-quences from the hybridizing probes reconstructs the com-plement of the original 12-mer target:

TCGGATCG

CGGATCGAGGATCGAC

GATCGACT

ATCGACTT

TCGGATCGACTT

Hybridization methodology can be carried out by attachingtarget DNA to a surface. The target is then interrogated witha set of oligonucleotide probes, one at a time (4, 5). Thisapproach can be implemented with well-established methodsof immobilization and hybridization detection but involves alarge number of manipulations. For example, to probe asequence utilizing a full set of octanucleotides, tens ofthousands of hybridization reactions must be performed.

Alternatively, SBH can be carried out by attaching probesto a surface in an array format where the identity ofthe probeat each site is known. The target DNA is then added to thearray of probes. The hybridization pattern determined in asingle experiment directly reveals the identity of all comple-mentary probes. The testing of this SBH format has requiredthe development of new technologies for the fabrication ofoligonucleotide arrays (6, 7). Most recently, an oligonucleo-tide array of 256 octanucleotides was generated using asolution-channeling device to direct the oligonucleotideprobe synthesis into 3 x 3 mm sites (7). In this format, theresolution limit of approximately 1 x 1 mm may ultimatelylimit the number of probes that one can synthesize on asubstrate of a manageable size (7).We report here a method by which light is used to direct the

synthesis of oligonucleotide probes in high-density, minia-turized arrays. Photolabile 5'-protected N-acyl-deoxynucle-oside phosphoramidites, surface linker chemistry, and ver-satile combinatorial synthesis strategies have been developedfor this technology. A matrix of 256 spatially defined oligo-nucleotide probes was generated, and the ability to use thearray to identify complementary sequences was demon-strated by hybridizing fluorescently labeled octanucleotides.The hybridization pattern demonstrates a high degree ofbasespecificity and reveals the sequence of oligonucleotide tar-gets.

MATERIALS AND METHODSThe basic strategy for light-directed oligonucleotide synthe-sis (6) is outlined in Fig. 1. The surface of a solid supportmodified with photolabile protecting groups (X) is illuminatedthrough a photolithographic mask, yielding reactive hydroxylgroups in the illuminated regions. A 3'-O-phosphoramidite-activated deoxynucleoside (protected at the 5'-hydroxyl witha photolabile group) is then presented to the surface andcoupling occurs at sites that were exposed to light. Followingcapping, and oxidation, the substrate is rinsed and the surfaceis illuminated through a second mask, to expose additionalhydroxyl groups for coupling. A second 5'-protected, 3'-O-phosphoramidite-activated deoxynucleoside is presented tothe surface. The selective photodeprotection and couplingcycles are repeated until the desired set of products is

Abbreviations: SBH, sequencing by hybridization; MeNPoc-,a-methyl-6-nitropiperonyloxycarbonyl-; DMT, 4,4'-dimethoxytritylchloride.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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hv

x xx x x

I I I I I

00,:r z/0

hv

x x xx x

I I I I I

T T O O O0

C-XZ7 4Z-IZ Z4k0

x x xI I

HO HO 0 0 0

x xx x xI I I I I4444C

T-X

x x x x xI I I I IT T o 0O

74S4o7 777777

C A T A TA G C T G

Ret T T C C G

Repeat 5 5 5 § &

FIG. 1. Light-directed synthesis ofoligonucleotides. A surface bearingphotoprotected hydroxyls (X-O) is il-luminated through a photolithographicmask (Mi), generating free hydroxylgroups in the photodeprotected re-gions. The hydroxyl groups are thencoupled to a deoxynucleoside phos-phoramidite (5'-photoprotected). Anew mask pattern (M2) is applied, anda second photoprotected phosphor-amidite is coupled. Rounds of illumi-nation and coupling are repeated untilthe desired set ofproducts is obtained.

obtained. Since photolithography is used, the process can beminiaturized to generate high-density arrays of oligonucleo-tide probes. Furthermore, the sequence of the oligonucleo-tides at each site is known.

5'-Photolabile N-AcyI-deoxynudeoside-3'-O-phosphwamid-ites. Two factors enter into the design of photolabile 5'-hydroxyl protecting groups. Because the bases have strongn-7* transitions in the 280-nm region, the deprotectionwavelength should be longer than 280 nm to avoid undesir-able nucleoside photochemistry. In addition, the photode-protection rates of the four deoxynucleosides should besimilar so that light will equally effect deprotection in differ-ent illuminated synthesis sites. To meet these criteria wehave developed a set of 5'-O-(a-methyl-6nitropiperonylox-ycarbonyl)-N-acyl-2'-deoxynucleosides (MeNPoc-N-acyl-deoxynucleosides) and measured their photokinetic behav-ior.The synthetic pathway for preparing 5'-O'-(a-methyl-6ni-

tropiperonyloxycarbonyl)-N-acyl-2'-deoxynucleoside phos-phoramidites (MeNPoc-N-acyl-2'-deoxynucleoside phos-phoramidites) is illustrated in Scheme I. In the first step, anN-acyl-2'-deoxynucleoside reacts with 1-(2-nitro-4,5-methylenedioxyphenyl)ethyl-l-chloroformate to yield 5'-MeNPoc-N-acyl-2'-deoxynucleoside. In the second step, the3'-hydroxyl reacts with 2-cyanoethyl N,N'-diisopropylchlo-rophosphoramidite using standard procedures to yield the5'-MeNPoc-N-acyl-2'-deoxynucleoside-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites. The photoprotectinggroup is stable under ordinary phosporamidite synthesis con-

H B

OH

NO2 Okc

\o

ditions and can be removed with aqueous base. These reagentscan be stored for long periods under argon at 4TC.A 0.1 mM solution of each of the four deoxynucleosides-

MeNPoc-dT, MeNPoc-dCibu, MeNPoc-dGPAC, and MeN-Poc-dAPAC-was prepared in dioxane. Aliquots (200 pl) wereirradiated with 14.5 mW of 365-nm light per cm2 in a narrow-path (2 mm) quartz cuvette for various times. Four or fivetime points were collected for each base, and the solutionswere analyzed for loss of starting material at 280 nm on aNucleosil 5-Cg HPLC column using a mobile phase of 60%6(vol/vol) in water containing 0.1% (vol/vol) trifluoroaceticacid [MeNPoc-dT required a mobile phase of 70%o (vol/vol)methanol in water]. Peak areas of the residual MeNPoc-N-acyl-deoxynucleoside were calculated, yielding photolysishalf-times of 28 s, 31 s, 27 s, and 18 s for MeNPoc-dT,MeNPoc-dCibu, MeNPoc-dGPAC, and MeNPoc-dAPAC, re-spectively. In all subsequent lithographic experiments, illu-mination times of 4.5 min (9 x ti/2MeNPoc-dC) were used toensure >99% removal of MeNPoc protecting groups.The synthesis support consists of a 5.1 x 7.6 cm glass

substrate prepared by cleaning in concentrated NaOH, fol-lowed by exhaustive rinsing in water. The surfaces were thenderivatized for 2 hr with a solution of 10%6 (vol/vol) bis(2-hydroxyethyl)aminopropyltriethoxysilane (Petrarch Chemi-cals, Bristol, PA) in 95% ethanol, rinsed thoroughly withethanol and ether, dried in vacuo at 400C, and heated at 1000Cfor 15 min. In these studies, a synthesis linker is attachedby reacting derivatized substrates with 4,4'-dimethoxytrityl(DMT)-hexaethyloxy-O-cyanoethyl phosphoramidite.

NO2 O

OH

0

0~~~~~P _'~C

B=APAc GPAC, CiBu, or T

Scheme I

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In a light-directed synthesis, the overall synthesis yielddepends on the photodeprotection yield, the photodeprotec-tion contrast, and the chemical coupling efficiency. Photo-kinetic conditions are chosen to ensure that photodeprotec-tion yields are >99%o. Unwanted photolysis in normally darkregions of the substrate can adversely affect the synthesisfidelity and can be minimized by using lithographic maskswith a high optical density (5 OD units) and by careful indexmatching of the optical surfaces.Two different methods were developed to investigate the

chemical coupling efficiencies of the photoprotected nucle-osides. First, the efficiencies were measured on hexaethyleneglycol derivatized control pore glass. The glycol linker wasdetritylated and a MeNPoc-deoxynucleoside-O-cyanoethylphosphoramidite coupled to the resin. Next, a DMT-deoxynucleoside-cyanoethyl phosphoramidite (reporter ami-dite) was added. The reporter amidite should couple to anyunreacted hydroxyl groups remaining from the first couplingreaction. Following DMT deprotection, the trityl effluentswere collected and quantified by absorption spectroscopy. Inthis assay, the coupling efficiencies are measured assuming ahigh coupling efficiency of the reporter amidite. The efficien-cies of the MeNPoc-deoxyribonucleoside-O-cyanoethylphosphoramidites to the hexaethylene glycol linker and the16 different dinucleotide efficiencies were measured. Thevalues ranged between 95% and 100%6 in this assay and wereindistinguishable from values obtained with standard DMT-deoxynucleoside phosporamidites.To measure the coupling efficiency of the photoprotected

nucleosides directly on the glass synthesis supports, each ofthe four MeNPoc-amidites was coupled to a substrate. Asection of the substrate was deprotected by illumination andallowed to react against a MeNPoc-phosphoramidite. A newregion of the substrate was then illuminated, a fluorescentdeoxynucleoside phosphoramidite (FAM-phosphoramidite;Applied Biosystems) was coupled, and the substrate wasscanned for fluorescence signal. Assuming that the fluores-cently labeled phosphoramidite reacts at both the newlyexposed hydroxyl groups and the previously unreacted hy-droxyl groups, then the ratio of fluorescence intensitiesbetween the two sites provides a measure of the couplingefficiency. This measurement of the surface chemical cou-pling yields efficencies ranging between 85% and 98%.

RESULTSSpatfally Directed Synthesis ofan Oligonucleotide Probe. To

initiate the synthesis of an oligonucleotide probe, MeNPoc-dCibu-3'-O-phosphoramidite was attached to a synthesis sup-port through a linker (hexaethyloxy-O-cyanoethyl phosphor-amidite). Regions of the support were activated for synthesisby illumination through 800 x 12800 ,m apertures of aphotolithographic mask. Seven additional phosphoramiditesynthesis cycles were performed (with DMT-protected de-oxynucleosides) to generate the sequence 3'-CGCATCCG.Following removal of the phosphate and exocyclic amineprotecting groups with concentrated NH4OH for 4 hr, thesubstrate was mounted in a water-jacketed thermostaticallycontrolled hybridization chamber.

Hybridization of Targets to Surface Oligonudeotides. Toexplore the availability of the support-bound octanucleotideprobes for hybridization, 10 nM 5'-GCGTAGGC-fluoresceinwas introduced into the hybridization chamber and allowedto incubate for 15 min at 15°C. The surface was then inter-rogated with an epifluorescence microscope (488-nm argonion excitation). The fluorescence image of this scan is shownin Fig. 2. The fluorescence intensity coincides with the 800 x12800 pm stripe used to direct the synthesis of the probe.Furthermore, the signal intensities are high (four times over

FIG. 2. Hybridization and thermal dissociation of oligonucleo-tides. Fluorescence scan of 5'-GCGTAGGC-fluorescein hybridizedto complementary probes. The substrate surface was scanned witha Zeiss Axioscop 20 microscope using 488-nm argon ion laserexcitation. The fluorescence emission above 520 nm was detectedusing a cooled photomultiplier (Hamamatsu 934-02) operated inphoton-counting mode. The signal intensity is indicated on the colorscale shown to the right of the image. The temperature is indicatedto the right of each panel in 'C.

the background of the glass substrate), demonstrating spe-cific binding of the target to the probe.The melting behavior of the target-probe complex was

investigated by increasing the temperature in the hybridiza-tion chamber. After 10 min of incubation at each tempera-ture, no significant changes in the fluorescence were ob-served, and the fluorescence intensities were recorded. Theduplex melted in the temperature range expected for thesequence under study. The probes were stable to temperaturedenaturation ofthe target-probe complex as demonstrated byrehybridization of target DNA.

Sequence Specificity of Target Hybridization. To demon-strate the sequence specificity of probe hybridization, twodifferent sequences were synthesized in 800 x 12800 pEmstripes. Fig. 3A identifies the location of the two probes. Theprobe 3'-CGCATCCG was synthesized in stripes 1, 3, and 5.The probe 3'-CGCTTCCG was synthesized in stripes 2, 4,and 6. Fig. 3B shows the results of hybridizing 5'-GCG-TAGGC-fluorescein to the substrate at 15°C. Although theprobes difer by only one internal base, the target hybridizesspecifically to its complementary sequence (-500 countsabove background in stripes 1, 3, and 5) with little or nodetectable signal in positions 2, 4, and 6 (-10 counts). Fig. 3Cshows the results of hybridization with targets to bothsequences. The signal in all positions in Fig. 3C illustratesthat the absence of signal in Fig. 3B is due solely to theinstability of the single base hybridization mismatch. Al-though the targets are present in equimolar concentrations,the ratios of signals in stripes 2, 4, and 6 in Fig. 3B areapproximately 1.6 times higher than the ratios of signals inregions 1, 3, and 5, presumably due to slight differences inmelting temperature values for the two duplexes. The du-plexes were dissociated by raising the temperature to 45°C for15 min, and the hybridizations were repeated in the reverse

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FiG. 3. Sequence specificity of hybridiza-tion. (A) Index ofthe probe composition at eachsynthesis site. 3'-CGCATCCG was synthesizedin stripes 1, 3, and 5, and 3'-CGCTTCCG wassynthesized in stripes 2, 4, 6. (B) Fluorescenceimage showing hybridization of substrate with10 nM 5'-GCGTAGGC-fluorescein. Hybridiza-tion was performed in 6x SSPE (1 M NaCl/66mM sodium phosphate/6 mM EDTA, pH 7.4)/0.1% Triton X-100 at 15TC for 15 min. (C)Hybridization with 10 nM 3'-GCGAAGGCadded to the hybridization solution of B. (D)After high-temperature dissociation of fluores-ceinated targets from C, the substrate was in-cubated with 10 nM 3'-GCGAAGCC at 15?C for15 min. (E) Hybridization with 10 aM 3'-GCGTAGGC added to the hybridization solu-tion of D.

order (Fig. 3 D and E), demonstrating specificity of hybrid-ization in the reverse direction.

Combinatorial Synthesis of a Probe Matrix. In a light-directed synthesis, the location and composition ofproductsdepend on the pattern of illumination and the order ofchemical coupling reagents (6). Consider the synthesis of256tetranucleotides, as illustrated in Fig. 4. Mask 1 activatesone-fourth of the substrate surface for coupling with the firstof 4 nucleosides in the first round of synthesis. In cycle 2,mask 2 activates a different quarter of the substrate forcoupling with the second nucleoside. The process is contin-ued to build four regions of mononucleotides. The masks ofround 2 are perpendicular to those of round 1, and each cyclegenerates four new dinucleotides. The process continuesthrough round 2 to form 16 dinucleotides as illustrated in Fig.4. The masks of round 3 further subdivide the synthesisregions so that each coupling cycle generates 16 timers. Thesubdivision of the substrate is continued through round 4 toform the tetranucleotides. The synthesis of this probe matrixcan be compactly represented in polynomial notation (6) as(A + C + G + TV'. Expansion of this reaction polynomialidentifies the 256 tetranucleotides.The potential of light-directed combinatorial synthesis can

be appreciated by the combinatorial synthesis of the probematrix shown in Fig. SA. The polynomial for this synthesis isgiven by 3'-CG(A + G + C +. TY4CG. The synthesis map isgiven in Fig. 5B. All possible tetranucleotides are synthesizedflanked by CG at the 3' and 5' ends. Hybridization of thetarget 5'-GCGGCGGC-fluorescein to this array at 15'C yields

T C G

1-

T

Round 1 Li -

TCG A

Round2T

TCG A

Round 3 - G

T C G A

c1TT

TCG A

T

the 3'-CGCCGCCG complementary probe as the most in-tense position (2698 counts). Significant intensity is alsoobserved for the following mismatches: 3'-CGCAGCCG (554counts), 3'-CGCCGACG (317 counts), 3'-CGCCGICG (272counts), 3'-CGACGCCG (242 counts), 3'-CGICGCCG (203counts), 3'-CGCCCCCG (180 counts), 3'-CGc(IGCCG (163counts), 3'-CGCCACCG (125 counts), and 3'-CGCCICCG(78 counts).

DISCUSSIONArrays of oligonucleotides can be efficiently generated bylight-directed synthesis and can be used to determine theidentity ofDNA target sequences. As shown here, an arrayof all tetranucleotides was produced in 16 cycles requiring 4hrto complete. Because combinatorial strategies are used (6),the number of compounds increases exponentially while thenumber of chemical coupling cycles increases only linearly.For example, expanding the synthesis to the complete set of4 (65,536) octanucleotides will add only 4 hr to the synthesisfor the 16 additional cycles. Furthermore, combinatorialsynthesis strategies can be implemented to generate arrays ofany desired composition (6). For example, since the entire setof dodecamers (412) can be produced in 48 photolysis andcoupling cycles (1, compounds requires b x n cycles), anysubset of the dodecamers (including any subset of shorteroligonucleotides) can be constructed with the correct litho-graphic mask design (6) in 48 or fewer chemical couplingsteps. The number of compounds in an array is limited only

T C GA

T C G AA

G

IT

T T T T................bird *lra]................

4iilllHlllllisitA....... .......

.............

'TTStllTlnll 11, ..1|[swX Em lX1 *]..............................And..................

44 tetranucleotides

FIG. 4. Combinatorial synthesis of44 tet-ranucleotides. In round 1, one-fourth of thesynthesis area is activated by illuminationthrough mask 1 for coupling of the firstMeNPoc-nucleoside (A in this case). In cy-cle 2 of round 1, mask 2 activates a differentone-quarter section of the synthesis sub-strate and a different nucleoside (T) is cou-pled. Further lithographic subdivisions ofthe array and chemical couplings generatethe complete set of 256 tetranucleotides asdescribed in the text.

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B

CG

GCGGCGGC-FlAOCTAOCTAGCT AOCT

r ...."... ".... .....T

I- -

I I I I I I I II I I I I I I Ic-lI IIIIII *I I I I lil

I I I I Aw- 1I Ia I T

Am~~~~~~~

lI I I I I1 lll| | § I § I § I X I 1 GIIIII I I I I -I 1

A

-I I ' 0

A

llI I [I I 1 I

T

I C

iG

A

CG

A G C TFIG. 5. Hybridization to a matrix of 256 octanucleotides. (A) Fluorescence image of a 256-octanucleotide aray following hybridization with

10 nM 5'-GCGGCGGC-fluorescein in 6x SSPE/0.1% Triton X-100 for 15 min at 150C. (B) Matrix decoder for the array. The matrix synthesisis represented by the polynomial 3'-CG(A + G + C + T)4CG. The site containing the complementary sequence 3'-CGCGCCCG is lightedfor reference.

by the density of synthesis sites and the overall array size.Recent experiments have demonstrated hybridization toprobes synthesized in 25-pm sites. At this resolution, theentire set of 65,536 octanucleotides can be placed in an arraymeasuring 0.64-cm square, and the set of 1,048,576 dodeca-nucleotides requires only a 2.56-cm array.

Genome sequencing projects will ultimately be limited byDNA sequencing technologies. Current sequencing method-ologies are highly reliant on complex procedures and requiresubstantial manual effort (8, 9). SBH has the potential fortransforming many of the manual efforts into more efficientand automated formats. Light-directed synthesis is an effi-cient means for enabling SBH and for the large-scale pro-duction of miniaturized arrays.

Oligonucleotide arrays are not limited to primary sequenc-ing applications. Because single base changes cause multiplechanges in the hybridization pattern, the oligonucleotidearrays will provide a powerful means to check the accuracyof previously elucidated DNA sequence or to scan forchanges within a sequence. In the case of octanucleotides, a

single base change in the target DNA results in the loss ofeight complements and generates eight new complements.Matching ofhybridization patterns may be useful in resolvingsequencing ambiguities from standard gel techniques or forrapidly detecting DNA mutational events.The potentially very high information content of light-

directed oligonucleotide arrays will change genetic diagnostictesting. Sequence comparisons of hundreds to thousands ofdifferent genes will be assayed simultaneously instead of thecurrent one, or few at a time format. Custom arrays can alsobe constructed to contain genetic markers for the rapididentification of a wide variety of pathogenic organisms.

Oligonucleotide arrays can also be applied to study thesequence specificity of RNA- or protein-DNA interactions.Experiments can be designed to elucidate specificity rules ofnon-Watson-Crick oligonucleotide structures or to investi-gate the use of novel synthetic nucleoside analogs for an-tisense or triple helix applications. Suitably protected RNAmonomers may be employed for RNA synthesis. The oligo-nucleotide arrays should find broad application deducing thethermodynamic and kinetic rules governing formation andstability of oligonucleotide complexes.

We are grateful to L. Stryer, P. Schultz, and F. Pease for theircritical reading and helpful comments on the manuscript. A.C.P. isa National Institutes of Health postdoctoral fellow (HG 00060). Thiswork was sponsored in part by National Institutes of Health (HG00813) and Department of Energy (DE FG03 92ER) grants toS.P.A.F.

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