A method for producing a high-density, position-addressable gene array is disclosed. The method includes contacting an array of different-sequence oligonucleotides having a unique, known combinatorial sequence associated with each position in the array with a set of extended gene probe templates which are complementary to the oligonucleotides at one of the template end regions. After hybridization, the oligonucleotides in the array are extended by strand-directed polymerization to form the probe array. Also disclosed is a probe array device formed by the method.

FIELD OF THE INVENTION 
The present invention relates to a method for forming position-addressable 
polynucleotide arrays, such as arrays of known-sequence genes or gene 
probes. 
REFERENCES 
Chu, T. J., et al., Electrophoresis 13:105-114 (1992). 
Coeling, K. J., in KIRK-OTHMER ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY, 3rd 
ed., John Wiley & Sons, New York, Vol. 6, 661-669. 
Fodor, S. P. A., et al., Science 251:767-773 (1991). 
Gait, M. J. , ed., OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, Oxford 
Univ. Press, 1990. 
Hames, B. D., et al., NUCLEIC ACID HYBRIDIZATION, IRL Press (1985). 
Lee, S. M., in ENCYCLOPEDIA OF POLYMER SCIENCE AND ENGINEERING, 2nd ed., 
Vol. 3, John Wiley & Sons, New York, 601-615. 
Matson, R. S. et al., Anal. Biochem. 217: 306-310 (1994). 
Matson, R. S. et al., Anal. Biochem. 224:110-116 (1995). 
Pirrung, et al., U.S. Pat. No. 5,143,854 (1992). 
Pirrung, M. C., et al., U.S. Pat. No. 5,143,854 
Sambrook, J., et al., in MOLECULAR CLONING: A LABORATORY MANUAL, 2nd 
Edition, Cold Spring Harbor Lab Press (1989). 
Southern, E., EP Patent No. 373,203 (1994). 
Southern, E. et al., Genomics 13:1008-1017 (1992). 
Virnekas, B., et al., Nuc. Acids Res. 22(25):5600 (1994). 
BACKGROUND OF THE INVENTION 
There is widespread interest in gene probe and gene arrays, for example, 
(i) for use in gene-based diagnostics aimed at detecting one of a number 
of possible mutations in a given gene, (ii) for identifying genomic or 
cDNA library species associated with one or more given RNA species, and 
(iii) for gene expression studies. 
Heretofore, arrays of genes, e.g., cDNAs or genomic library clones, have 
been formed by spotting individual genes on suitable substrates, e.g., 
nitrocellulose filter paper, for subsequent blotting, e.g., with 
radiolabeled probes. This approach is relatively labor-intensive, 
requiring that each gene be individually spotted on the substrate. The 
approach is also limited in array-region density to the physical 
resolution achievable by the device used for spotting. 
More recently, a method for preparation of high density 
position-addressable oligomer arrays on a planar substrate has been 
reported (Fodor, Pirrung). In this method a substrate having 
photoprotective groups is irradiated in selected regions only, using 
photolithographic mask techniques, to deprotect surface active groups in 
those selected regions. The entire surface is then treated with a solution 
of a selected subunit, which itself has a photoprotected group, to react 
this subunit with the surface groups in the photodeprotected regions. This 
process is repeated to (i) add a selected subunit at each region of the 
surface, and (ii) build up different-sequence oligomers at known, 
addressable regions of the surface. 
This method has the advantage that reaction sites do not have to be 
physically separated during subunit addition, and therefore massive 
parallel subunit addition is possible by applying subunit-addition 
reagents over the entire surface of the array. Greater site density is 
therefore feasible than in systems where physical separation of reagents 
is required from one reaction site to another, and where individual 
reagents are spotted or deposited in defined array regions. 
Co-owned patent application Ser. No. 08/512,027 for Method and Apparatus 
for Producing Position-Addressable Combinatorial Libraries, filed Aug. 7, 
1995, discloses another method for producing position-addressable, 
high-density arrays of oligomers. The approach involves forming a 
one-dimensional position-addressable array of oligomers on a filament, by 
a series of subunit addition reactions in which each of a plurality of 
subunits is reacted with different segments of the filament wound on a 
spool, where successive subunit reactions are carried out on successively 
smaller-diameter (or larger-diameter) spools. 
Like the two-dimensional photo-masking techniques, the one-dimensional 
filament-spool method just outline employs massive parallel subunit 
synthesis to efficiently produce high-density arrays of 
position-addressable oligomers, e.g., oligonucleotides. Thus, for example, 
to form a hexamer array of 4.sup.6 oligonucleotides, only four addition 
reactions are required at each subunit addition step (one for each of the 
four nucleotides), so that the total 4.sup.6 array can be produced in 
4.times.6=24 reactions. By contrast, if each subunit were added separately 
to each array region, a total of 4.sup.6 separate subunit addition steps 
would be required (as, for example, proposed by Southern). 
The direct use of massive parallel subunit addition, for synthesis of 
position-addressable oligomer libraries, is not readily adaptable to the 
synthesis of position-addressable gene arrays for two reasons. First, 
since the gene sequences are expected to be random rather than 
combinatorial, there is no simple way of patterning the genes on a 
substrate so that the unique gene sequences can be built up by massive 
parallel step-wise synthesis. Secondly, good gene-sequence fidelity would 
be obtainable in high-density arrays only up to about 6-10 subunits, 
whereas genes or gene probes of interest will typically contain 15-100 or 
more nucleotides. 
It would therefore be useful to provide a method of forming high-density, 
position-addressable arrays of genes or gene probes which can be produced 
in a way that takes advantage of the massive parallel synthesis methods 
just outlined, but which is not constrained by the limitations just 
discussed. 
SUMMARY OF THE INVENTION 
The invention includes, in one aspect, a method of producing a 
position-addressable array of known-sequence gene probes. The method 
employs an array of different-sequence oligonucleotides, where each 
oligonucleotide in the array has a unique, known combinatorial sequence 
associated with a known-address region in the array. This array is 
preferably a combinatorial oligonucleotide array of the type described 
above, produced by massive parallel subunit addition methods. 
The array is contacted with a set of gene-probe templates, where each 
member of the set has a probe segment whose sequence is complementary to a 
selected, known-sequence gene probe and a recognition segment whose 
nucleotide sequence is complementary to one of the array oligonucleotides. 
The contacting is carried out under complementary-strand hybridization 
conditions, such that each member in the template set becomes hybridized, 
through its recognition segment, to a complementary-sequence 
oligonucleotide in the array of oligonucleotides. 
The oligonucleotide probes in the array are then extended, by 
strand-directed polymerization, along the probe segments of the templates 
hybridized to the oligonucleotides, to produce the desired probe array. 
The probe segments in the set of gene-probe templates are preferably at 
least 10-15 nucleotide bases in length, and may be up to several hundred 
bases. The oligonucleotides forming the array library preferably form a 
combinatorial library of different-sequence oligonucleotides having a 
selected length of typically 3 and 8 subunits. 
In one general embodiment, the position-addressable array of 
different-sequence oligonucleotides is formed on a wound or extended 
filament having a one-dimensional array of regions, each carrying a 
different-sequence oligonucleotide in said combinatorial library. The 
linear density of the oligonucleotides on the filaments may be 100/cm or 
greater. 
In another general embodiment, the position-addressable array of 
different-sequence oligonucleotides is formed on a planar substrate having 
a two-dimensional array of regions, each carrying a different-sequence 
oligonucleotide in said combinatorial library. The density of 
oligonucleotides on the array is preferably 1000/cm.sup.2. 
For use in constructing a position-addressable array of long, typically 
biological gene probes, e.g. cDNAs, cloned genomic fragments, and 
expressed sequence tags (ESTs), the method may further include the steps 
of contacting the probe array formed above with a second set of gene-probe 
templates whose members each have a probe-recognition segment whose 
sequence is complementary to a selected, known-sequence gene probe segment 
in the existing probe array, and a gene segment which is complementary to 
a selected gene sequence. The contacting is carried out under 
complementary-strand hybridization conditions such that each member in the 
second set of templates becomes hybridized, through its probe-recognition 
segment, to a complementary-sequence gene probe in the probe array. 
The oligonucleotides in the probe array are then extended, by 
strand-directed polymerization, along the extended gene segment of 
templates hybridized to the oligonucleotides, to produce the desired 
gene-probe array. 
In another aspect, the invention includes a position-addressable gene array 
device formed in accordance with this method. The device includes a 
substrate having a linear or planar array of regions, and a 
different-sequence gene probe attached to each array region, through a 
known-sequence oligonucleotide segment which has a unique, known, 
combinatorial sequence associated with a given array region. The 
oligonucleotide segments in the array preferably form a combinatorial 
library of different-sequence oligonucleotides having a selected length of 
between 3 and 10 subunits, and the different-sequence gene probes 
preferably include at least 10, and up to several hundred or more 
nucleotide subunits. 
As above, the position-addressable array of different-sequence 
oligonucleotide segments may be formed on a wound or extended filament 
having a one-dimensional array of regions, each carrying a 
different-sequence oligonucleotide segment, preferably at a linear density 
of different-sequence segments of at least about 100/cm. 
Alternatively, the array of different-sequence oligonucleotide segments may 
be formed on a planar substrate having a two-dimensional array of regions, 
each carrying a different-sequence oligonucleotide segment, and at a 
preferred density of at least 1000/cm.sup.2. 
These and other objects and features of the invention will become more 
fully apparent when the following detailed description of the invention is 
read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
I. Definitions 
The terms below have the following meanings, unless indicated otherwise: 
"Oligonucleotides" refers to nucleotide oligomers nucleotides containing 
typically between about 3 and 50 nucleotide subunits. In the context of 
oligonucleotides attached at their 5' ends to an array support, in a 
position-addressable oligonucleotide array, the subunits forming the 
oligonucleotide may include or be composed primarily of nucleotide analog 
subunits, or other subunits capable of forming sequence-specific 
Watson-Crick base pairing, when assembled in a linear polymer, with the 
proviso that the free ends of the oligonucleotides are ribonucleotide or 
deoxyribonucleotide subunits capable of providing a suitable substrate for 
strand-directed polymerization in the presence of a DNA polymerase and one 
or more nucleotide triphosphates, e.g., conventional deoxyribonucleotides 
with free 3' 0H groups. 
A "known-sequence oligonucleotide" is an oligonucleotide whose base 
sequence in known. 
A "gene probe" is an oligonucleotide or polynucleotide which has a base 
sequence complementary to that of a region of a gene, genomic fragment, 
cDNA, messenger RNA, expressed sequence tag (EST) or other known-sequence 
nucleic acid, where the region of complementarity is typically between 10 
to 100 or more bases, and the gene probe itself is preferably 10-50 or 
more bases. 
A "position-addressable array of different-sequence oligonucleotides or 
probes" refers to a linear or planar array of oligonucleotides, each 
oligonucleotide having a different, known, unique sequence associated with 
a known location (address) in the array. 
A "combinatorial library of oligonucleotides" is a set of oligonucleotides 
containing substantially each sequence permutation that can be formed by 
placing a selected one of a number of different subunits at each of a 
selected number of residue positions. These residue positions may be 
contiguous or may be interrupted by one of more residues filled with a 
single subunit (or subunit sequence) only. 
A "combinatorial sequence" refers to one of the possible permutation 
sequences in a combinatorial library of oligonucleotides. 
A "high-density array" of oligonucleotides, probes, or gene fragments 
(regions) refers to a linear array of at least 100 regions/cm, or to a 
planar array of at least 1,000 regions/cm.sup.2. 
"Complementary-strand hybridization conditions" refer to temperature, ionic 
strength and/or solvent conditions effective to produce sequence-specific 
hybridization between an single-stranded oligonucleotide and its 
complement-sequence nucleic acid strand. Such conditions are preferably 
stringent enough to prevent hybridization of two nearly-complementary 
strands that have one or more internal base mismatches. 
"Strand-directed polymerization" refers to nucleic acid strand extension, 
in the presence of a suitable DNA or RNA polymerase, and all four 
deoxynucleotide triphosphates, of primer strand hybridized to a template 
strand, where the sequence of bases in the template strand directs the 
sequence of bases added in the primer strand. 
II. Producing Position-Addressable Oligonucleotide Arrays 
This first section describes linear and planar oligonucleotide arrays 
useful in practicing the invention. The linear array is formed according 
to the steps detailed in co-owned U.S. patent application Ser. No. 
08/512,027 for Method and Apparatus for Producing Position-Addressable 
Combinatorial Libraries, filed Aug. 7, 1995 and incorporated herein by 
reference. The method is outlined below with respect to FIGS. 1-8. A 
planar oligonucleotide array, such as the array shown fragmentarily in 
FIG. 9, may be constructed according to known methods (e.g., Pirrung, 
Southern). 
Considering the construction of a high-density linear array of 
oligonucleotides, FIG. 1 shows a spool 20 having a square cross-section 
defining four equal axially extending surface regions, such as regions 22, 
24. A filament 26 wound on the spool has multiple windings, such as 
windings 28, each encircling the spool one time. The spool partitions each 
winding into four equal segments, such as segments 30 extending across the 
upper surface of the spool in the figure, and segments 32 extending across 
an adjacent spool surface. It will be appreciated that the segments 
disposed over any given spool surface region, e.g., segments 30, are 
regularly spaced segments of the filament, with such in an extended, 
linear form. In the present case, such segments have a length W 
corresponding to the width of each surface region on the spool, and are 
separated by 3.times.W length intervals. 
Each group of segments is reacted sequentially with one of a plurality of 
selected reagents, to chemically attach a selected nucleotide subunit to 
the segments in each surface region, such as discussed below with respect 
to FIGS. 8A-8D. 
After the above reaction steps, the filament is wound on a second spool 
having a plurality of axially extending surface regions whose widths are 
preferably four times that of the first spool, or one-quarter that of the 
previous spool, if he stepwise subunit addition is performed on 
successively smaller spools (see below). The filament winding operations 
and spool-surface reaction steps are continued until the desired 
oligonucleotide library is formed. 
To determine the number and sizes of spools to be used, it is useful first 
to determine the total number of subunit addition steps required and the 
order in which the addition steps will be carried out. As an example, it 
will be assumed that the desired library is to be formed by a series of 
six subunit-position addition steps, where the total number of 
different-sequence compounds in the library will be 4.sup.6, or 4,096. 
Four separate reagents will be used per subunit addition step; therefore, 
four-sided spools will be used. The final (smallest) spool containing this 
library will have 4,096/4=1,024 windings. Assuming a filament thickness of 
15 microns, and a 5 micron inter-winding spacing, the total displacement 
of the wound filament upon the spool will be 1,024.times.20 microns, or 
about 2 cm. 
In order to limit the total filament length, it is desirable to place these 
1,024 windings on a spool having a small side dimension, but sufficient to 
prevent spool bending over the spool length, e.g., 2 cm, of the windings. 
In the present case, a metal spool, e.g., stainless steel spool, having a 
square I-beam or crossbar configuration, and a side dimension of 0.5 mm is 
selected. The total length of the filament used is then 1,024.times.0.5 
mm.times.4 (sides), or about 2 m. The final library density will be about 
4.times.10.sup.3 /2m, or about 200/cm. 
Table 1 below shows the diameters of the spools used in constructing a 
library having this filament length and thickness and numbers of filament 
windings, starting with the smallest spool. The last synthesis step, 
involving attaching four nucleotides to four 0.5 m segments of the 
filament, does not involve reaction on a spool. 
TABLE 1 
______________________________________ 
Step # of Members Width per Side 
______________________________________ 
1 4 500 microns 
2 16 2 mm 
3 64 8 mm 
4 256 3.2 cm 
5 1,024 12.8 cm 
6 4,096 approx. 0.5 m 
(not wound) 
______________________________________ 
The filament employed must be of a material that, at dimensions in the 15 
micron range, will be highly flexible and of high tensile strength to 
prevent stretching during filament winding on different-size spools. In 
one preferred embodiment, the filament is a metal wire, typically 2-25 
microns in diameter, coated with a thin suitable polymer coating. e.g., 
0.5-2 micron coating thickness. Suitable wires of these dimensions formed 
of copper, platinum, platinum-iridium alloy (U.S. Pat. No. 5,201,903), or 
stainless steel are known. 
Suitable polymers for the coating are polystyrene, polypropylene, nylon, 
polyacrylamide, PVC, and the like. Methods for forming thin polymer 
coatings, e.g., 0.5-2 microns coating thickness, on a metal substrate are 
known (Lee, Coeling, and U.S. Pat. Nos. 5,201,903 and 5,137,780). Such 
methods include spray coating (Coeling), vacuum deposition and various 
methods of in situ polymerization directly onto the wire (Lee). Such 
methods of in situ polymerization include glow discharge polymerization, 
electron beam polymerization, and photopolymerization, the last of which 
has been used to form ultrathin coatings of &lt;0.1 micron. 
Preferably, the polymer is applied to the wire in an uncrosslinked state 
and is then irradiated to lightly crosslink the resin coating, thus 
reducing swelling during the reaction steps of the method. Irradiation 
crosslinking is primarily effective with polyolefins, such as polystyrene, 
polybutadiene, and polypropylene. The use of functionalized polystyrene 
resins for solid state synthesis is well established, and polypropylene 
has been reported as a useful substrate for oligonucleotide synthesis 
(Matson, et al.). 
For purposes of illustrating the method, the reaction order will be from 
smaller to larger spools. In actual practice, it is generally preferred to 
carry out the reactions in the reverse order, so that the final reaction 
is carried out on the smallest spool, which then forms the library device 
that can be used for preparing gene probe arrays in accordance with the 
invention (Section III). 
FIGS. 2A and 2B illustrate attachment of the first sequence of nucleotide 
bases to the filament, where the features in the figure have the same 
numbers as in FIG. 1. Linkage sites on each filament winding segment are 
indicated by tick marks, such as marks 34 on segment 32. Although the tick 
marks are shown on one side of each winding segment, it is understood that 
the linkage sites are present on all sides of the filament segments. 
As will be seen below, these linkage sites preferably include a linker used 
in attaching the growing oligonucleotide chain to the filament, where the 
linker itself may include at its free end, a fixed-sequence 
oligonucleotide segment, such as described below with respect to FIGS. 10 
and 11. This pre-library sequence is used to optimize probe hybridization 
specificity and stability with a library sequence, as discussed below. 
As a first step in the synthesis of the library oligonucleotide, the 
filament segments associated with region 22 in the spool are reacted with 
a reagent solution of a 3'-protected adenosine nucleoside, which is added 
to the channel, indicated by 36, formed by region 22, and closed at 
opposite spool ends by end plates, such as plate 38 seen in FIG. 1. 
Although the reagent is shown added to the level of the winding segments in 
the figures, it will be understood that the reagent solution is preferably 
added to a level forming a positive meniscus, such that all sides of the 
windings segments are bathed in the solution, and this consideration 
applies to all of the subunit or substituent addition reactions described 
below. 
The reaction is effective to add the selected nucleoside, e.g. 
deoxyadenosine (A), to the filament at the linkage sites, where such sites 
include a linker designed for reacting with the unprotected 5'-OH group of 
the added nucleotide, for example, where the linker terminates in a 3' 
phosphoramidate activated nucleoside. 
The subunit addition reaction is then repeated successively for each of the 
three remaining reaction regions, to successively attach the selected 
nucleosides deoxyguanosine (G, closed circles), deoxycytidine (C, closed 
squares) and deoxythymidine (T, open squares) to each of different groups 
of filament segments, as indicated in FIG. 2B. The resulting filament 
contains the sequence of nucleosides shown in FIG. 3, where each 
nucleotide is attached to periodically recurring segments of length L (the 
width of a spool side), and the four nucleosides form a repeating sequence 
of length 4L on the filament. 
As will be seen in FIG. 8, the newly added nucleosides are 3'-protected 
during the linkage step. The protecting groups must be removed and the 
3'-0H group activated to enable reaction with the next series of 5'-free 
OH nucleotide subunits. The deprotection and activation steps may be 
carried out with the filaments still wound on the first spool, with the 
filament unwound, or after winding the filament on the second spool. 
With reference to FIGS. 4A and 4B, the filament is now wound on a second 
spool 40, which, in the illustrated example, has a width 4L, corresponding 
to the length of the repeating sequence of four nucleosides in FIG. 3. For 
example, from the spool sizes given in Table 1, the first spool may have a 
width L of 500 microns, and the second spool a width 4L, or 2 mm. The 
number of windings on the second spool is consequently 1/4 the number of 
first-spool windings, and the displacement of the windings on the spool 
may be proportionately less. 
As above, spool 40 defines four channels, such as channels 42 and 44, which 
are used to hold the reagents used in attaching the second series of 
nucleotide subunits to the filament segments. As shown in FIG. 4A, each 
complete sequence formed in the first-spool reaction steps is contained in 
each of a multiplicity of segments associated with a single region, such 
as region 46, disposed over a single side on the second spool. As 
illustrated in FIGS. 4A and 4B, the 3'-activated segments in each region 
are successively exposed to reaction with one of the four 3'-protected 
nucleoside reagents, to add each of these nucleosides to the segments in 
each region, as indicated, following the general reactions steps discussed 
above. 
The resulting filament has the nucleotide sequence shown in FIG. 5. Each 
repeating sequence is now of length 16L and consists of all possible 
permutations (4.sup.2) of dimers based on the four nucleotide bases. 
The filament is then wound on a third spool 48, which, in the illustrated 
example, has a width 16L, corresponding to the length of the repeating 
sequence of 16 nucleotide dimers in FIG. 5, e.g., 8 mm in Table 1. Again, 
each complete sequence formed in the previous reaction is contained in the 
multiplicity of filament segments disposed over each region, such as 
region 50, defined by each face of the third spool. 
After removal of the 3'-protecting groups in the terminal subunits, and 
activation of these 3'-groups, the segments disposed over each region are 
successively exposed to a reactive solution of each of the four 
3'-protected nucleosides, as described above. The reactions are effective 
to add each nucleoside to each of the 16 dimers, represented by X's in 
FIGS. 5 and 6A-6B, attached to the filament segments associated with each 
region, such as region 50 in FIG. 6A. 
The resulting filament has the nucleotide sequence which is partially shown 
in FIG. 7. Each repeating sequence is now of length 64L and consists of 
all possible permutations (4.sup.3) of trimers based on the four 
nucleotide bases. FIG. 7 primarily illustrates the portion of this 
sequence, 16L in length, which has A as its third nucleotide. It will be 
appreciated that repeating groups of 16 dimers will have each of the four 
different nucleotides at the third position, collectively forming the 64 
different trimer sequences that repeat themselves along the length of the 
filament. 
These steps are repeated with the increasing-size spools, such as the two 
additional spools indicated in Table 1. In the present example, this leads 
to a 4.sup.5 -member library, consisting of all possible permutations of 
pentamers based on the four nucleotide bases. 
The final step, leading to a 4.sup.6 -member library, would involve only a 
single winding, since the extended filament at this stage contains four 
repeating sequences of pentamers and thus would be wound only once around 
a spool whose side width would be about 0.5 m. It may be more convenient 
to carry out this final subunit addition by (i) removing the 5'-protecting 
groups from the chain-end subunits and (ii) immersing each of the four 
segments of the filament, each containing a complete sequence of 
pentamers, into one of four solutions of activated, 5'-protected 
nucleotide subunits (A,C,G, and T, as in the previous steps). 
The resulting filament will contain a single sequence of hexamers making up 
a 4.sup.6 -member library and containing all possible permutations of 
hexamers based on the four nucleotide bases. According to an important 
feature of the method, the sequence at each position along the filament, 
whether in linear or wound form, is known from the steps employed in 
library synthesis. By way of example, if the 4.sup.6 -member 
oligonucleotide library above is constructed in a larger-to-smaller spool 
direction, and always in the order of subunit addition A, G, C, T, the 
first quarter segment of the filament position would contain A at the 
first subunit position, the second quarter segment, G at this position, 
and so on. Within each of these quarter segments, the first quarter 
subsegment would contain an A at the second subunit position, the second 
quarter subsegment, G at the second subunit position, and so on. At the 
final smallest-spool, the final (sixth) subunit added would of course be 
the particular subunit added at each of the spool's surface regions. 
FIG. 8 shows one exemplary synthetic scheme used for preparation of the 
library of oligonucleotides on a resin-coated filament, in accordance with 
the invention. In FIGS. 8A-8D, the filament is indicated fragmentarily at 
51, and includes a metal wire core 52 encased in a thin polymer coating 
54. 
The polymer coating is derivatized, conventionally, with a linker 56, to 
which the first nucleotide subunit (or a pre-library subunit) is then 
attached via its 5' OH end. The linker molecules are preferably of 
sufficient length to permit the compounds in the completed library device 
to interact freely with probe sequences to which the device is exposed in 
forming the probe array of Section III. Longer linkers are also known to 
lead to more efficient nucleoside coupling reactions (Gait, p. 45). 
The linkage in the present example may be formed by (i) reacting the coated 
polymer, e.g., a chloromethylated polystyrene, or chlorinated 
polypropylene (Matson; Chu) with a long chain bifunctional reagent such as 
a diol, diamine, ethylene glycol oligomer or amine-terminated ethylene 
glycol oligomer; and (ii) reaction of the free hydroxyl or amino end of 
the linker with the first nucleoside (or a pre-library nucleotide or 
sequence), whose 5'-hydroxyl has been converted to a suitable leaving 
group such as a mesylate, and which is protected in its 3' position with a 
suitable protecting group, e.g., phenoxyacetyl (Pac) (Virnekas) and also 
base-protected. After coupling to the linker, the 3' OH group of the newly 
added nucleotide is then deprotected, e.g., by reaction with NH.sub.3 in 
MeOH, and activated with MeOP(N.sup.i Pr.sub.2)Cl in EtN.sup.i Pr.sub.2, 
using standard procedures (Virnekas), and as illustrated in FIGS. 8A and 
8B. 
The phosphotriester will be converted to a phosphate linkage after 
oligonucleotide synthesis is complete. Also, the 
substrate-to-oligonucleotide linkage is base stable, and the 
oligonucleotides will thus remain bound to the substrate throughout the 
deprotection steps which conclude the synthesis. This issue has been 
addressed by Southern and Matson, inter alia. 
After 3'-deprotection and activation at the 3' 0H group, a second 
3'-protected nucleoside (e.g., deoxyguanosine) is added, giving the dimer 
AG sequence (FIG. 8C) after oxidation. These steps are repeated with 
further nucleoside units (FIGS. 8C and 8D) until the desired 
oligonucleotides have been formed on the filament. At this point, the 
terminal 3'-hydroxy groups are deprotected, and activated, as above, 
except that the terminal nucleotide is left in a free 3'-0H form. Finally, 
the methyl groups on the phosphotriester linkages are removed by treatment 
with thiophenol or ammonia, and the purine and pyrimidine bases are 
deprotected, e.g., by treatment with ammonia, all according to known 
methods (e.g., Gait). 
Each nucleoside added in the synthesis is 3'-protected, preferably by a 
phenoxyacetyl (Pac) group. The exocyclic amino groups on the purine and 
pyrimidine bases of the nucleosides are also protected, as amides, 
throughout the sequence, according to well established methods (Gait), and 
can be deprotected by treatment with ammonia upon completion of the 
library synthesis. Because the coupling reactions are sensitive to air and 
moisture, they are preferably carried out under an inert atmosphere. 
The members of the oligonucleotide library illustrated above consist of a 
sequence of single deoxyribonucleotides. Alternatively, the subunits 
forming the library may be dinucleotides, trinucleotides, or higher order 
oligonucleotides. For example, at each subunit addition step, one of 
typically 4-20 different trinucleotide "subunits" corresponding to one of 
up to amino acid codons, could be added at each subunit addition step 
(Virnekas). 
FIG. 9 shows a fragmentary portion of a position-addressable planar array 
60, having a support substrate 62 defining a plurality of discreet array 
regions, such as regions 64, 66. Each region supports attached molecules 
of an oligonucleotide having a known sequence, and the oligonucleotides 
collectively form a combinatorial library of nucleotide sequences, each 
being associated with a known address or region of the array. 
The oligonucleotides on the array are preferably 3-10 nucleotides subunits 
in length (where "subunit" refers to single or multiple-nucleotide units), 
and are attached to the substrate through a linker, similar to the 
attachment of oligonucleotides to a filament substrate. Methods of forming 
high-density oligonucleotide arrays of this type have been described, 
e.g., Pirrung, U.S. Pat. No. 5,143,854 and Southern, EP Patent No. 373,203 
(1994). The step-wise nucleotide addition reactions described in these 
references may have to be modified for synthesis in a 5' to 3' direction, 
employing, for example, the approach discussed with reference to FIG. 8. 
III. Producing Position-Addressable Probe and Gene Arrays 
In forming a position-addressable array of gene probes, in accordance with 
the present invention, a position-addressable array of the 
oligonucleotides described above is contacted with a set of gene-probe 
templates, where each template set has a probe segment whose sequence is 
complementary to a selected, known-sequence gene probe and a recognition 
segment whose nucleotide sequence is complementary to one of the 
oligonucleotides in the array. This contacting is carried out under 
complementary-strand hybridization conditions such that each template 
becomes hybridized, through its recognition segment, to a 
complementary-sequence oligonucleotide in the array of oligonucleotides. 
The oligonucleotides in the array are then extended, by strand-directed 
polymerization, along the probe segments of the templates hybridized to 
the oligonucleotides, to produce the desired probe array. 
FIG. 10 shows a fragmentary cross sectional portion of a spool 68 and a 
segment 70 of a filament in a spooled filament of the type shown in FIG. 
1. The filament segment shown includes two sides 70a, 70b of a four-sided 
winding, where each of the four sides of each winding has attached 
combinatorial oligonucleotides which differ from one another in one 
sequence position--in this case, in the terminal 3' position. The 
oligonucleotide molecules O.sub.n in side 70a in the figure include three 
fixed "pre-library" sequences 5'-TTA-3', which are provided on a linker in 
the filament, prior to library sequence construction, and six nucleotides 
5'-ACCGGC-3' which form one of the 4096 possible six subunit permutations 
of four single nucleotides. Similarly, the oligonucleotide molecules 
O.sub.n+1 attached to the side 70b of the segment include the three fixed 
"pre-library" sequences and six nucleotides 5'-ACCGGT-3' which form 
another one of the 4096 possible six subunit permutations of the 
oligonucleotide library. 
The purpose of three pre-library sequences is to provide a total of at 
least 9-10 nucleotides in the oligonucleotides for hybridization with 
complementary regions of a gene-probe template, thus improving the 
specificity of the hybridization and increasing the temperature at which 
base-specific hybrids will stably form (e.g., Hames, and Sambrook, 11.8). 
These advantages may also be achieved by using, for example, di- or 
trinucleotides as the library subunits, so that a six-subunit library 
contains 12 or 18 nucleotides, respectively. 
Two members of a set of gene-probe templates are shown at 72, 74 in FIG. 
11, and designated P.sub.n and P.sub.n+1, respectively. Each template in 
this set, such as template P.sub.n, contains a 3' nine-nucleotide 
recognition segment 72a whose nucleotide sequence is complementary to one 
of the oligonucleotides in the array--in this case oligonucleotide O.sub.n 
--and a twelve-nucleotide probe segment 74b whose sequence is 
complementary to a selected, known-sequence 12mer gene probe to be 
included in the array. The second template shown, P.sub.n+1, similarly has 
a recognition sequence that is complementary to O.sub.a+1, and a probe 
segment that is complementary to a second, different gene probe to be 
included in the array. 
The set of templates can be synthesized by conventional solid state 
oligonucleotide synthesis methods (Gait), where each member of the set is 
individually synthesized and purified, then combined in preferably 
equimolar amounts to form the template set. 
For a 4096-oligonucleotide array, the template set may contain up to 4096 
different gene-probe segments, although fewer total gene sequences, with 
some gene-sequence duplications at selected library positions may be 
desirable, e.g., for internal control. 
The templates are contacted, i.e., placed in solution in the presence of, 
the oligonucleotides of the array, under conditions that lead to 
hybridization between the recognition segments of the templates and 
complementary array oligonuclotides. The hybridization conditions are 
preferably stringent (high-criterion) conditions in which only hybrids 
with a high degree of homology form. Typical high criterion conditions are 
about 8.degree. C. lower than the melting temperature T.sub.m, (Hames, p. 
108). Thus, for example, in the case of 9mer hybrids, where a melt 
temperature of about 40.degree. C. may be expected, a high-criterion 
annealing or hybridization temperature may be about 32.degree. C. 
FIGS. 12A-12D illustrate the steps in forming a probe array in accordance 
with the invention, where the figure numbers and the oligonucleotide 
sequences designated O.sub.n, O.sub.n+1, P.sub.n, and P.sub.n+1, are the 
same as in FIGS. 10 and 11. Contacting an oligonucleotide array (FIG. 12A) 
with a set of gene-probe templates under suitable hybridization 
conditions, as above, leads to sequence-specific attachment of the probes 
in the set to position-addressable oligomers in the array, as illustrated 
in FIG. 12B, with the gene-probe segment in each probe extending beyond 
the 3' end of the associated array oligomer. The oligonucleotides attached 
to the array are now extended by strand-directed polymerization along the 
associated gene segments, to produce the double-stranded gene probes 
indicated in FIG. 12C. Suitable polymerization conditions in the presence 
of a DNA polymerase, e.g., Klenow fragment of E. coli DNA polymerase I, T4 
DNA polymerase, T7 polymerase, and all four nucleotide triphosphates 
(NTP's), are well known (e.g., Sambrook, 5.35-5.51). 
In general, under the high-criterion hybridization conditions that can be 
selected, any internal base mismatch will prevent formation of a stable 
duplex. Further, a mismatch at the 3' terminus of the oligonucleotide may 
block successful strand-directed polymerization, thus limiting strand 
extension to exact basepair matches between the array oligonucleotides and 
template recognition segments. 
As a final step, the double-stranded probes in the array may be denatured, 
e.g., by heating above the T.sub.m of the probe, and the template strand 
released, as indicated in FIG. 12. The final probe array, indicated at 76 
in FIG. 12D, includes a substrate spool 68, having a linear array of 
regions, such as regions 70a, 70b, where each of the regions has a 
different-sequence gene probe fragment attached thereto. The gene probes 
are attached to each region through an oligonucleotide segment 
(corresponding to the original array oligonucleotide), where these 
segments have a unique, known, combinatorial sequence of nucleotide 
subunits associated with a known-position array region, and collectively 
form a combinatorial library of sequence permutations. The gene-probe 
segments in the array are at least 10 bases in length, and typically, 
15-50 bases. 
In the gene-probe embodiment just described, the set of template 
oligonuclotides employed are readily synthesized by solid-phase methods. 
Such methods may be difficult to apply, however, where the gene-probe 
segments correspond to genomic fragments, cDNA's, EST's or other genetic 
coding regions with lengths of 50 to several hundred or more bases. In 
this case, it is advantageous to employ biological synthesis for producing 
the long gene probe fragments, and rely on relatively short synthetic 
oligonucleotides for coupling the long segments to the array. This 
approach is illustrated in FIGS. 13A-13D. 
FIG. 13A shows a fragmentary cross-sectional portion of the gene-probe 
array shown in FIG. 12D, where the gene-probes in the array, indicated at 
P.sub.n and P.sub.n+1, have the sequences shown in FIG. 11. In this case 
the probe segment in each region of the array is complementary in base 
sequence to the 3'-end region sequence in one of a set of large gene-probe 
templates, such as those designated F.sub.n and F.sub.n+1. As already 
indicated, the large gene probes are preferably produced biologically, 
typically as closed genomic, cDNA or EST fragments. The 3'-end region of 
the large gene-probe templates that are complementary to the gene-probe 
segments on the already-formed gene probe array are also referred to 
herein as recognition sequences. Preferably these are 15 bases or more in 
length, to provide good hybridization specificity with the array gene 
probes. 
Thus, to practice this embodiment of the invention, it is only necessary to 
know the base sequence of each recognition segment in the large gene-probe 
templates, although typically the sequences of large contiguous regions of 
100 or more bases will be known. 
The method for forming a long gene-probe arrays closely follows that 
discussed above with respect to FIGS. 12A-12D. Briefly, a set of long 
gene-probe templates, each having a recognition segment complementary to 
the probe segment of an oligonucleotide probe array is contacted with the 
array, under preferably high-criterion hybridization conditions. Where, 
for example, the array-probe segment has 20 bases of complementarity with 
the recognition segment of the gene-probe templates, and an average 
T.sub.m, of about 60.degree.-70.degree. C., the hybridization temperature 
should be 52.degree.-62.degree. C. 
The contacting step is effective to hybridize each long gene probe 
template, e.g. F.sub.n and F.sub.n+1, with a corresponding known-position 
probe recognition sequence, as indicated in FIG. 13B. These templates now 
provide for strand-directed polymerization in the presence of a suitable 
polymerase and NTP's, as above, to form the double-strand long gene probes 
shown in FIG. 13C. As a final step, the array have treated to remove 
template strands, as above. 
The resulting probe array, indicated at 78 in FIG. 13D, includes a 
substrate spool 68, having a linear array of regions, such as regions 70a, 
70b, where each of the regions has a different-sequence gene probe--in 
this case, of up to several hundred bases in length--attached to that 
region through an oligonucleotide segment (corresponding to the original 
array oligonucleotide), where these segments have a unique, known, 
combinatorial sequence of nucleotide subunits associated with a 
known-position array region, and collectively form a combinatorial library 
of sequence permutations. 
IV. Utility 
The gene probe arrays of the invention have a variety of uses in 
sequencing-by-hybridization (SBH), diagnostics, and gene-expression 
studies. 
As an example in the diagnostics area, the gene probes in the array can 
represent sequences corresponding to a number of different known mutations 
in a selected gene, e.g., the cystic fibrosis gene, where each different 
mutation is associated with a known addressable region of the array. 
To test for a particular mutation in a DNA sample, a DNA sample is first 
labeled, e.g., with a fluorescent label, then reacted with the probes in 
the array under high-criterion hybridization conditions. Examination of 
the array, e.g., by fluorescence microscopy, is then used to identify gene 
probes where binding has occurred. Knowing the position of the labeled 
array region, the exact mutation in the sample can be determined. A large 
array can of course be designed to carry multiple gene types, each having 
several different possible mutations. 
In the area of gene expression studies, labeled cDNA's from a cell or cell 
type in a given state can be reacted with a large cDNA-library array 
prepared as above, to determine which of the cell genes are being 
expressed and at what levels, for purposes of, for example, (i) monitoring 
changes in gene expression during a treatment, or (ii) identifying library 
cDNA's which are associated with changes in cell state or cell type. 
Although the invention has been described with respect to particular spool 
structures, methods, libraries, and library devices, it will be 
appreciated that various changes and modification can be made without 
departing from the invention.