Method and apparatus for conducting an array of chemical reactions on a support surface

The invention provides apparatus and methods for making arrays of functionalized binding sites on a support surface. The invention further provides apparatus and methods for sequencing oligonucleotides and for identifying the amino acid sequence of peptides that bind to biologically active macromolecules, by specifically binding biologically active macromolecules to arrays of peptides or peptide mimetics.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to methods for conducting a large number of chemical 
reactions on a support surface, methods for making the support surface, 
and the support surface itself. 
2. Summary of the Related Art 
Proposals for the direct sequencing of DNA by hybridization with arrays of 
oligonucleotides are known in the art. Drmanac et al., Genomics 4; 114 
(1989) proposes hybridization array-mediated DNA sequencing by binding 
target DNA to a dot blot membrane, followed by probing with an array of 
oligonucleotides. Khrapko et al., FEBS Letters 256, 118 (1989) proposes 
hybridization array-mediated DNA sequencing by binding the oligonucleotide 
array to a support membrane, followed by probing with target DNA. 
Synthesis of arrays of bound oligonucleotides or peptides is also known in 
the art. Houghton, in the Multiple Peptide System product brochure 
describes the T-bag method, in which an array of beads is physically 
sorted after each interaction. This method becomes unwieldy for the 
preparation of large arrays of oligonucleotides. Geysen et al., J. 
Immunol. Methods 102; 259 (1987) discloses the pin method for the 
preparation of peptide arrays. The density of arrays that may be produced 
by this method is limited, and the dipping procedure employed in the 
method is cumbersome in practice. Southern, Genome Mapping and Sequencing 
Conference, May 1991, Cold Spring Harbor, N.Y., disclosed a scheme for 
oligonucleotide array synthesis in which selected areas on a glass plate 
are physically masked and the desired chemical reaction is carried out on 
the unmasked portion of the plate. In this method it is necessary to 
remove old mask and apply a new one after each interaction. Fodor et al., 
Science 251; 767 (1991) describes a method for synthesizing very dense 50 
micron arrays of peptides (and potentially oligonucleotides) using 
mask-directed photochemical deprotection of synthetic intermediates. This 
method is limited by the slow rate of photochemical deprotection and by 
the susceptibility to side reactions (e.g., thymidine dimer formation) in 
oligonucleotide synthesis. Khrapko et al, FEBS Letters 256; 118 (1989) 
suggests simplified synthesis and immobilization of multiple 
oligonucleotides by direct synthesis on a two dimensional support, using a 
printer-like device capable of sampling each of the four nucleotides into 
given dots on the matrix. However, no particulars about how to make or use 
such a device are provided. 
Some methods for permanently attaching oligonucleotides to glass plates in 
a manner suitable for oligonucleotide synthesis are known in the art. 
Souther, Chem. abst. 113; 152979r (1990) describes a stable phosphate 
ester linkage for permanent attachment of oligonucleotides to a glass 
surface. Mandenius et al., Anal. Biochem. 157; 283 (1986) teaches that the 
hydroxyalkyl group resembles the 5'-hydroxyl of oligonucleotides and 
provides a stable anchor on which to initiate solid phase synthesis. 
The related art contains numerous ideas and information related to arrays 
of chemical reactants on a solid support. However, existing or suggested 
methods are limited, and do not conveniently and reliably produce the very 
large, high density arrays. There is, therefore, a need for new methods 
for preparing large high density arrays of reactive sites. Ideally, such 
methods should utilized relatively simple machinery to produce large, 
dense arrays of solid phase bound reactants in a reproducible and rapid 
manner. 
SUMMARY OF THE INVENTION 
This invention provides a method for conducting a large number of chemical 
reactions on a support surface. Solutions of chemical reactants are added 
to functionalized binding sites on the support surface by means of a 
piezoelectric pump. This pump deposits microdroplets of chemical reactant 
solution onto the binding sites. The chemical reactant at each binding 
site is separated from the others by surface tension. Typically, the 
support surface has 10-10.sup.4 functionalized binding sites per cm.sup.2 
and each functionalized binding site is about 50-2000 microns in diameter. 
Typically, the amounts of reagents added to each binding site is in a 
volume of about 50 picoliter to 2 microliter. The reactions at the 
functionalized binding site may form covalent bonds such as esters or 
amide bonds or may involve non-covalent specific binding reactions such as 
antibody/antigen binding or oligonucleotide specific binding. The 
invention also includes array plates and methods for making the array 
plates. 
Typically, the array plates are made by the process set out in FIG. 2A by 
(a) coating a support surface with a positive or negative photoresist 
substance which is subsequently exposed and developed to create a 
patterned region of a first exposed support surface; 
(b) reacting the first support surface with a fluoroalkylsilane to form a 
stable fluoroalkylsiloxane hydrophobic matrix on the first support 
surface; 
(c) removing the remaining photoresist to expose a second support surface; 
and 
(d) reacting the second support with a hydroxy or aminoalkylsilane to form 
derivatized hydrophilic binding site regions. 
The preferred siloxane reaction product of the present invention is 
tetradecafluoro-1,1,2,2-tetrahydrooctyl siloxane. In FIG. 2A, the hatched 
lines are the solid support, "Si" represents a first exposed support 
surface site, "Si--F" is a hydrophobic fluoroalkylsilane site, and 
"Si--OH" is a derivatized hydrophilic binding site. 
Alternatively, the array plates can be made by the process set out in FIG. 
2B by 
(a) reacting a support surface with a hydroxy or aminoalkylsilane to form a 
derivatized hydrophilic support surface; 
(b) reacting the support surface form step (a) with o-nitrobenzyl carbonyl 
chloride as a temporary photolabile blocking to provide a photoblocked 
support surface; 
(c) exposing the photoblocked support surface of step (b) to light through 
a mask to create unblocked areas on the support surface with unblocked 
hydroxy or aminoalkylsilane; 
(d) reacting the exposed surface of step (c) with perfluoroalkanoyl halide 
or perfluoroalkylsulfonyl halide to form a stable hydrophobic 
(perfluoroacyl or perfluoroalkylsulfonamido) alkyl siloxane matrix; and 
(e) exposing this remaining photoblocked support surface to create 
patterned regions of the unblocked hydroxy- or aminoalkylsilane to form 
the derivatized hydrophilic binding site regions. 
The preferred siloxanes of the present invention are 3-perfluorooctanoyloxy 
propylsiloxane and 3-perfluorooctanesulfonamido propylsiloxane. In FIG. 
2B, the hatched lines are the solid support, "--A" represents a 
hydrophilic support site, "--A B" represents a temporary photolabile 
blocked support site, and "--A F" represents a hydrophobic site. 
The invention also provides a method for determining or confirming the 
nucleotide sequence of a target nucleic acid. The target nucleic acid is 
labelled by conventional methods and hybridized to an oligonucleotides of 
known sequence previously bound to sites on the array plate. The array 
plate having bound labelled target nucleic acid is then washed at 
appropriate stringency and the presence and location of bound labelled 
target nucleic acid is determined using scanning analyzers. Since the 
sequence of the covalently attached oligonucleotide in each element on the 
array is known, this allows the unambiguous determination of the 
nucleotide sequence of the target nucleic acid. 
The methods of the invention may also be applied to the determination of 
peptides or peptide mimetics that bind biologically active receptors. In 
this aspect, peptide arrays of known sequence can be applied to glass 
plates using the same piezoelectric pump/surface tension wall method 
described supra. The resulting array of peptides can then be used in 
binding analyses with biologically active receptor ligands to screen for 
peptide mimetics of receptor agonists and antagonists. Thus, the invention 
provides a method for producing peptide array plates, peptide array plates 
having covalently bound peptides separated by surface tension areas, and 
methods of using such peptide array plates to screen for peptide mimetics 
of receptor agonists and antagonists. 
Those skilled in this art will recognize a wide variety of binding site and 
chemical reactants for forming either covalent bonds or for specific 
binding reagents.

DETAILED DESCRIPTION OF THE INVENTION 
The practice of present invention can include a number of photoresist 
substances. These substances are readily known to those of skill in the 
art. For example, an optical positive photoresist substance (e.g., AZ 1350 
(Novolac.TM. type-Hoechst Celanese.TM.) (Novolac.TM. is a proprietary 
novolak resin, which is the reaction product of phenols with formaldehyde 
in an acid condensation medium)) or an E-beam positive photoresist 
substance (e.g., EB-9 (polymethacrylate by Hoya.TM.)) can be used. 
A number of siloxane functionalizing reagents can be used, for example: 
1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with diborane, 
and H.sub.2 O.sub.2 to oxidize the alcohol) 
a. allyl trichlorochlorosilane.fwdarw..fwdarw.3-hydroxypropyl 
b. 7-oct-1-enyl trichlorochlorosilane.fwdarw..fwdarw.8-hydroxyoctyl 
2. Diol (dihydroxyalkyl) siloxanes (silylate surface, and hydrolyze to 
diol) 
a. glycidyl trimethoxysilane.fwdarw..fwdarw.(2,3-dihydroxypropyloxy)propyl 
3. Aminoalkyl siloxanes (amines require no intermediate functionalizing 
step) 
a. 3-aminopropyl trimethoxysilane.fwdarw.3-aminopropyl 
4. Dimeric secondary aminoalkyl siloxanes 
a. bis (3-trimethoxysilylpropyl) amine.fwdarw.bis(silyloxylpropyl)amine 
In addition, a number of alternative functionalized surfaces can be used in 
the present invention. These include the following: 
1. Polyethylene/polypropylene functionalized by gamma irradiation or 
chromic acid oxidation, and reduction to hydroxyalkyl surface. 
2. Highly crosslinked polystyrene-divinylbenzene derivatized by 
chloromethylation, and aminated to benzylamine functional surface. 
3. Nylon--the terminal aminohexyl groups are directly reactive. 
4. Etched, reduced polytetrafluoroethylene. 
There are two important characteristics of the masked surfaces in patterned 
oligonucleotide synthesis. First, the masked surface must be inert to the 
conditions of ordinary oligonucleotide synthesis; the solid surface must 
present no free hydroxy, amino or carboxyl groups to the bulk solvent 
interface. Second, the surface must be poorly wet by common organic 
solvents such as acetonitrile and the glycol ethers, relative to the more 
polar fuctionalized binding sites. 
The wetting phenomenon is a measure of the surface tension or attractive 
forces between molecules at a solid-liquid interface, and is defined in 
dynes/cm.sup.2. Fluorocarbons have very low surface tension because of the 
unique polarity (electronegativity) of the carbon-flourine bond. In 
tightly structured Langmuir-Blodgett type films, surface tension of a 
layer is primarily determined by the percent of fluorine in the terminus 
of the alkyl chains. For tightly ordered films, a single terminal 
trifluoromethyl group will render a surface nearly as lipophobic as a 
perfluoroalkyl layer. When fluorocarbons are covalently attached to an 
underlying derivatized solid (highly crosslinked polymeric) support, the 
density of reactive sites will generally be lower than Langmuir-Blodgett 
and group density. However, the use of perfluoroalkyl masking agents 
preserves a relatively high fluorine content in the solvent accessible 
region of the supporting surface. 
There are also two important characteristics of the derivatized regions in 
patterned oligonucleotide synthesis. The surface must be compatible with 
the method of detection of hybridization. Radioactivity is largely being 
replaced by spectroscopic, chemiluminescent and fluorescent detection 
techniques in DNA research. It is desirable that the surface be optically 
transparent. A second important characteristic is that the linkage of the 
penultimate oligonucleotide to the surface have high chemical stability, 
at least equal to that of the polyphosphate backbone in DNA. 
The optical properties of glass (polytetrasiloxane) are unsurpassed for 
detection purposes. Further, there are numerous techniques developed by 
the semiconductor industry using thick films (1-5 microns) of photoresists 
to generate masked patterns of exposed glass surfaces. The best method to 
derivatize the first exposed glass surface is with volatile fluoroalkyl 
silanes using gas phase diffusion to create closely packed lipophobic 
monolayers. The polymerized photoresist provides an effectively 
impermeable barrier to the gaseous fluoroalkyl silane during the time 
period of derivatization of the exposed region. Following lipophobic 
derivatization however, the remaining photoresist can be readily removed 
by dissolution in warm organic solvents (methyl, isobutyl, ketone, or 
N-methyl pyrrolidone) to expose a second surface of raw glass, while 
leaving the first applied silane layer intact. This second region glass 
can then be derivatized by either solution or gas phase methods with a 
second, polar silane which contains either a hydroxyl or amino group 
suitable for anchoring solid phase oligonucleotide synthesis. 
Siloxanes have somewhat limited stability under strongly alkaline 
conditions. Conditions such as 0.1N sodium hydroxide, typically employed 
to strip probes from nylon hybridization membranes, should be avoided for 
reusable glass based hybridization arrays. 
Teflon (polytetrafluoroethylene) itself would provide an ideal lipophobic 
surface. Patterned derivatization of this type of material can be 
accomplished by reactive ion or plasma etching through a physical mask or 
using an electron beam, followed by reduction to surface hydroxymethyl 
groups. However, the opacity of teflon at visible wavelengths severely 
restrict the applicable methods for detection of hybridization. 
Depending on the ultimate application, other organic polymers have 
desirable characteristics for patterned oligonucleotide synthesis. 
Polypropylene is relatively transparent to visible light. It can be 
surface derivatized by chromic acid oxidation, and converted to hydroxy- 
or aminomethylated surfaces which provide oligonucleotide synthesis 
anchors of high chemical stability. Highly crosslinked 
polystryene-divinylbenzene (ca. 50%) is non-swellable, and can be readily 
surface derivatized by chloromethlylation and subsequent functional group 
manipulation. Nylon provides an initial surface of hexylamino groups. 
The lipophobic patterning of these surfaces can be effected using the same 
type of solution based thin film masking techniques and gas phase 
derivatization as glass, or by direct photochemical patterning using 
o-nitrobenzylcarbonyl blocking groups. Perfluoroalkyl carboxylic and 
sulfonic acid derivatives rather than silanes are now used to provide the 
lipophobic mask of the underlying surface during oligonucleotide 
synthesis. 
The solution of chemical reactant can be added to the functionalized 
binding site through utilization of a piezoelectric pump (FIG. 5) in an 
amount where the solution of chemical reactant at each binding site is 
separate from the solution of chemical reactant at other binding sites by 
surface tension. As described more fully infra, in the pump depicted in 
FIG. 5, reactant solution is inserted through the inlet (2) into the 
chamber (6) formed between the upper (1) and lower (5) plates of the 
piezo. Application of a voltage difference across the upper and lower 
plates causes compression of the piezo, forcing a microdroplet (4) out 
through the nozzle (3). 
FIG. 3 depicts the deposition of the reactant solution on a functionalized 
binding site and subsequent reaction with the surface. A micro-droplet of 
solution (FIG. 3(a)) is deposited on the functionalized binding site 
(center cross-hatched region in FIG. 3(b)). Because of the differences in 
wetting properties of the reactant solution on the functionalized binding 
site and the surrounding surface, the micro-droplet of the reactant 
solution beads on the functionalized binding site and the reactants in 
solution react with the surface (FIG. 3(c)). 
The piezoelectric pump that may be utilized in the invention delivers 
minute droplets of liquid to a surface in a very precise manner. The pump 
design is similar to the pumps used in ink jet printing. The picopump is 
capable of producing 50 micron or 65 picoliter droplets at up to 3000 Hz 
and can accurately hit a 250 micron target in a 900.degree. C. oven at a 
distance of 2 cm in a draft free environment. Preferred embodiments of the 
apparatus according to the invention are set forth in Example 3. 
Alternative pump designs should take into account the following physical 
and mechanical considerations for reliable performance to be obtained. 
When a non-compressible fluid inside of a pumping cavity is subjected to a 
rapid strong pressure pulse, the direction of flow of the liquid from the 
cavity is determined primarily by the inertial resistance of the liquid 
displaced. There is more liquid, and thus resistance to flow, on the inlet 
side than through the nozzle port. The column of liquid that is forced out 
of the nozzle begins to neck off as a result of surface tension. The 
stream breaks as the piezoelectric is de-energized, with the remaining 
column of liquid drawn back into the nozzle. The droplet that has necked 
off continues its flight with the velocity it achieved in the initial 
acceleration. Typically, the ejection velocity is about 1-2 meters/sec. 
In normal printing applications using 150 micron drops of viscous 
water-based inks, the head speed is typically about 0.5 meter/sec. This 
motion adds a transverse velocity component to the droplet trajectory and 
can affect aiming accuracy. It may also cause the drop to skip when it 
hits a surface. Droplets fired from a stationary head tend to evaporate 
more slowly because they follow in the vapor trail of the preceding drop. 
The heads work most reliably when the inlet supply lines are not required 
to flex and the liquids are not subjected to acceleration forces. 
The size of the drop is determined primarily by the surface tension of the 
solution and by the diameter of the pump nozzle. The smaller the droplet, 
the faster it will evaporate and the more its trajectory will be affected 
by drafts. Nozzles smaller than 25 microns tend to become plugged with 
dust particles. For water, the drop diameter is approximately 1.5 times 
the nozzle diameter. Typically, drops will not vary in size by more than 
5%. We have shown that the jet will also successfully eject a variety of 
polar solvents, including CH.sub.3 CN and MeOH. With these less viscous 
solvents, too forceful an ejection pulse may result in the formation of a 
series of trailing satellite droplets in addition to the primary drop. The 
duration of the pulse also affect satelliting. 
After the cavity has returned to its original state, a period of time must 
be allowed for the nozzle to refill by capillary action before another 
cycle of pulsing can be initiated. It is important for the nozzle refill 
only to the top of the orifice, but the liquid meniscus not spread out 
onto the front face of the jet. This is prevented by silanizing the face 
to reduce its surface tension. The head is also operated under slight 
negative pressure to prevent overfilling. The aim of the drop is in the 
axial direction of the nozzle, but defects in the face coating can affect 
the trajectory. 
Arrays of nozzles with up to 64 independent pumping chambers but a common 
inlet supply have been fabricated. It is important that each chamber inlet 
have some restriction so that operation of one pumping chamber does not 
affect the others. The separation between nozzles is typically 400 microns 
for printing applications, but denser arrays can be produced either by 
interleaving the transverse motion of the target or decreasing the nozzle 
spacing. 
EXAMPLE 1 
Preparation of Array Plates Ready for Oligonucleotide or Peptide Assembly 
The hybridization array is synthesized on a glass plate. The plate is first 
coated with the stable fluorosiloxane 3-(1,1-dihydroperfluoroctyloxy) 
propyltriethoxysilane. A CO.sub.2 laser is used to ablate off regions of 
the fluorosiloxane and expose the underlying silicon dioxide glass. The 
plate is then coated with glycidyloxypropyl trimethoxysilane, which reacts 
only on the exposed regions of the glass to form a glycidyl epoxide. The 
plate is next treated with hexaethyleneglycol and sulfuric acid to convert 
the glycidyl epoxide into a hydroxyalkyl group, which acts as a linker 
arm. The hydroxyalkyl group resembles the 5'-hydroxide of nucleotides and 
provides a stable anchor on which to initiate solid phase synthesis. The 
hydroxyalkyl linker arm provides an average distance of 3-4 nm between the 
oligonucleotide and the glass surface. The siloxane linkage to the glass 
is completely stable to all acidic and basic deblocking conditions 
typically used in oligonucleotide or peptide synthesis. This scheme for 
preparing array plates is illustrated in FIGS. 2(A) and 2(B) and was 
previously discussed. 
EXAMPLE 2 
Assembly of Oligonucleotides on the Array Plates 
The hydroxyalkylsiloxane surface in the dots has a surface tension of 
approximately .gamma.=47, whereas the fluoroxysilane has a surface tension 
of .gamma.=18. For oligonucleotide assembly, the solvents of choice are 
acetonitrile, which has a surface tension of .gamma.=29, and diethylglycol 
dimethyl ether. The hydroxyalkylsiloxane surface is thus completely wet by 
acetonitrile, while the fluorosiloxane masked surface between the dots is 
very poorly wet by acetonitrile. Droplets of oligonucleotide synthesis 
reagents in acetonitrile are applied to the dot surfaces and tend to bead 
up, as shown in FIG. 3. Mixing between adjacent dots is prevented by the 
very hydrophobic barrier of the mask. The contact angle for acetonitrile 
at the mask-dot interface is approximately .theta.=43.degree.. The plate 
effectively acts as an array microliter dish, wherein the individual wells 
are defined by surface tension rather than gravity. The volume of a 40 
micron droplet is 33 picoliter. The maximum volume retained by a 50 micron 
dot is approximately 100 picoliter, or about 3 droplets. A 100 micron dot 
retains approximately 400 picoliter, or about 12 droplets. At maximum 
loading, 50 micron and 100 micron dots bind about 0.07 and 0.27 femtomoles 
oligonucleotide, respectively. 
Assembly of oligonucleotides on the prepared dots (FIG. 2B, bottom) is 
carried out according to the H-phosphonate procedure (FIG. 4), or by the 
phosphoroamidite method. Both methods are well known to those of ordinary 
skill in the art. Oligonucleotide and Analogs, A Practical Approach (F. 
Eckstein ed., 1991). Delivery of the appropriate blocked nucleotides and 
activating agents in acetonitrile is directed to individual dots using the 
picopump apparatus described in Example 3. All other steps, (e.g., DMT 
deblocking, washing) are performed on the array in a batch process by 
flooding the surface with the appropriate reagents. An eight nozzle 
piezoelectric pump head is used to deliver the blocked nucleotides and 
activating reagents to the individual dots, and delivering droplets at 
1000 Hz, requires only 32 seconds to lay down a 512.times.512 (262k) 
array. Since none of the coupling steps have critical time requirements, 
the difference in reaction time between the first and last droplet applied 
is insignificant. 
EXAMPLE 3 
Construction of Piezoelectric Impulse Jet Pump Apparatus 
Piezoelectric impulse jets are fabricated from Photoceram (Corning Glass, 
Corning, N.Y.), a UV sensitive ceramic, using standard photolithographic 
techniques to produce the pump details. The ceramic is fired to convert it 
to a glassy state. The resulting blank is then etched by hydrogen 
fluoride, which acts faster in exposed then in nonexposed areas. After the 
cavity and nozzle details are lapped to the appropriate thickness in one 
plate, the completed chamber is formed by diffusion bonding a second (top) 
plate to the first plate. The nozzle face is lapped flat and surface 
treated, then the piezoelectric element is epoxied to the outside of the 
pumping chamber. When the piezoelectric element is energized it deforms 
the cavity much like a one-sided bellows, as shown in FIG. 5. 
To determine the appropriate orifice size for accurate firing of 
acetonitrile droplets, a jet head with a series of decreasing orifice 
sizes is prepared and tested. A 40 micron nozzle produces droplets of 
about 65 picoliter. 
A separate nozzle array head is provided for each of the four nucleotides 
and a fifth head is provided to deliver the activating reagent for 
coupling. The five heads are stacked together with a mechanically defined 
spacing. Each head has an array of eight nozzles with a separation of 400 
microns. 
The completed pump unit is assembled with the heads held stationary and the 
droplets fired downward at a moving array plate as shown in FIG. 6. The 
completed pump unit assembly (3) consists of nozzle array heads (4-7) for 
each of the four nucleotidase and a fifth head (8) for activating reagent. 
When energized, a microdroplet (9) is ejected from the pump nozzle and 
deposited on the array plate (1) at a functionalized binding site (2). 
A plate holding the target array is held in a mechanical stage and is 
indexed in the X and Y planes beneath the heads by a synchronous screw 
drives. The mechanical stage is similar to those used in small milling 
machines, microscopes and microtomes, and provides reproducible 
positioning accuracy better than 2.5 microns or 0.1 mil. As shown in FIG. 
7, the plate holder (3) is fitted with a slotted spacer (4) which permits 
a cover plate (5) to be slid over the array (6) to form an enclosed 
chamber. Peripheral inlet (1) and outlet (2) ports are provided to allow 
the plate to be flooded for washing, application of reagents for a common 
array reaction, or blowing the plate dry for the next dot array 
application cycle. 
Both the stage and head assembly are enclosed in a glove box which can be 
evacuated or purged with argon to maintain anhydrous conditions. With the 
plate holder slid out of the way, the inlet lines to the heads can be 
pressurized for positive displacement priming of the head chambers or 
flushing with clean solvent. During operation, the reagent vials are 
maintained at the ambient pressure of the box. 
With a six minute chemistry cycle time, the apparatus can produce 10-mer 
array plates at the rate of 1 plate or 10.sup.6 oligonucleotides per hour. 
EXAMPLE 4 
Use of Oligonucleotide Array Plates to Determine the Nucleotide Sequence of 
a Target Nucleic Acid 
The oligonucleotide array plate is prepared as described in Examples 1 and 
2, using the apparatus described in Example 3. The array contains 
oligonucleotides having 10 nucleotides each (10-mers). The synthesis is 
carried out such that each oligonucleotide element, moving in a 5'-3' 
direction, is identical to the preceding element in nucleotide sequence, 
except that it deletes the 5'-most nucleotide, and adds a new 3'-most 
oligonucleotide. In this way the total array represents every possible 
permutation of the 10-mer oligonucleotide. Oligonucleotides are spaced at 
7 nm intervals to provide an oligonucleotide loading density of 
3.4.times.10.sup.-12 moles/cm.sup.2, or 2.6.times.10.sup.-16 moles per 100 
micron element. The target nucleic acid is used to probe the 
oligonucleotide array plate. The probe is labelled with 1000 Ci/nmol 
P.sup.32. The labelled probe is contacted with the oligonucleotide array 
plate for hybridization in a 10 nM solution of probe in 3M Me.sub.4 NCl at 
42.degree. C. At 10% hybridization and wash efficiency, each 
oligonucleotide element dot having an exact match with the probe binds 26 
attomoles of probe. Radiolabel binding is detected using a Bio-Image 
Analyzer.TM. (Fuji, Waltham, Mass.). The pattern of binding is assessed 
and the nucleotide sequence of the probe nucleic acid is determined by 
ordering the nucleotide sequence according to the known sequences of the 
oligonucleotide elements, as shown in FIG. 1. 
FIG. 1 depicts a sequencing arrangement based on a matrix of trimer 
oligonucleotides bound to the array plate. FIG. 1(a) is the basic matrix 
consisting of the four nucleotides. FIG. 1(b) is the complete trimer 
matrix, representing each of the 4.sup.3 trimer permutations. The 
underlined elements in the array represent sites to which the target 
nucleic acid is bound. FIG. 1(c) depicts how a sequence complementary to 
the target nucleic acid is constructed from the known sequences of the 
sites to which the target nucleic acid is bound. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATTCTTGTTA10