Method of generating nucleic acid oligomers of known composition

The present invention is directed to a method for providing oligonucleotides or oligonucleotide analogs having known subunit sequences in which the desired oligomers are released from selected storage sites in one, two, or three dimensions, on a substrate by locally denaturing double-stranded complexes at the storage sites containing the desired oligomers. The released oligomers are useful in schemes for determining solutions to mathematical problems, in methods wherein hybridizing oligomers are used to encrypt and transmit data, in diagnostic and screening assay methodologies, and as primers or building blocks for synthesizing larger polynucleotides.

FIELD OF THE INVENTION 
This invention pertains to a method for providing oligomers of known 
subunit sequence which hybridize specifically to DNA and RNA having 
complementary nucleotide sequences, in which the desired oligomers are 
released from selected storage sites on a substrate by locally denaturing 
double-stranded complexes at the storage sites containing the desired 
oligomers. The released oligomers are oligonucleotides or oligonucleotide 
analogs, and are useful in schemes for determining solutions to 
mathematical problems, in methods wherein hybridizing oligomers are used 
to encrypt and transmit data, in diagnostic and screening assay 
methodologies, and as primers or building blocks for synthesizing larger 
polynucleotides. The present invention also features providing oligomers 
having desired subunit sequences from a device comprising a substrate 
supporting an array of oligomer-storing depot sites made by a novel method 
for the synthesis of DNA arrays which utilizes local melting of hybridized 
DNA and produces a set of substrate-attached oligomers of known subunit 
sequence. The present invention has applications in the fields of 
molecular computation, biochemistry, molecular biology, pharmacology, 
medical diagnostic technology, and data encryption and transmission. 
BACKGROUND OF THE INVENTION 
All publications and patent applications herein are incorporated by 
reference, fully as if each individual publication or patent application 
was specifically and individually indicated to be incorporated by 
reference. 
Various strategies for finding solutions to mathematical problems have been 
devised which use sets of DNA oligonucleotides having selected length and 
sequence properties. For example, DNA-based methods are developed for 
solving a Hamiltonian path problem (Adleman, Science, 1994, Vol. 266, 
pages 1021-3), a "satisfaction" problem (Lipton, Science, 1995, Vol. 268, 
pages 542-5), and for performing addition (Guarnieri et al., 1996, 
Science, vol. 273, pages 220-223) and matrix multiplication (Oliver, J. 
Molecular Evolution, 1997, Vol. 45, pages 161-7 ) of non-negative numbers. 
Each computation requires a set of oligonucleotides having properties 
tailored to the problem to be solved. Thus, a rapid and efficient method 
for providing custom sets of oligonucleotides having selected sequence and 
length properties is essential for efficient application of DNA-based 
computation methods. 
The present ability to detect oligonucleotides that are bound in a 
sequence-specific manner to discrete sites of a hybridization array 
permits the use of oligonucleotides to encrypt and transmit data; a use 
which, like nucleic acid computation, requires numerous custom sets of 
oligonucleotides having particular sequences and hybridization properties. 
Oligonucleotides are also used as hybridization probes to detect specific 
nucleic acid sequences in DNA and RNA samples immobilized on a variety of 
filter and solid supports, as in DNA and RNA Dot, Southern, and Northern 
blots, and in colony and plaque hybridization assays. These methodologies 
are widely used in the isolation and cloning of specific nucleic acids, 
and the diagnosis of disease caused by pathogens and genetic mutations 
(Berent et al., BioTechniques, issue of May/June 1985, pages 208-20; and 
J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning, A 
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring 
Harbor, New York, 1989, Chapter 11). After detection of labeled probes on 
a hybridization filter, it is a common practice to expose the 
hybridization filter to denaturing conditions such as solution of low 
ionic strength and high temperature, in order to wash the hybridizing 
probe molecules from the filter, making the filter ready for 
re-hybridization with a different hybridization probe (Protocols for DNA 
and RNA Transfer, DNA Electrotransfer, and Protein Transfer to Biodyne A 
Nylon Membranes, Pall Ultrafine Filtration Corporation, East Hills, N.Y., 
1985, page 14). 
Sets of oligonucleotides of defined sequence are used as primers for 
polymerases in polynucleotide synthesis and in nucleic acid amplification, 
for example, by the polymerase chain reaction (PCR, see Erlich, PCR 
Technology, Stockton Press, New York, 1989, in entirety). Sets of 
oligonucleotides of defined sequence are also used as probes of 
macromolecular structure, and are screened to identify oligomers which, 
either as antisense or as triplex-forming oligonucleotides, bind 
specifically to a native target nucleic acid such as a folded mRNA 
molecule (see, for example, Milner et al., Nature Biotechnology, 1987, 
Vol. 15, pages 537-41; and U.S. Pat. No. 5,176,996). 
More recently, oligonucleotides have been immobilized or synthesized in 
micro-arrays on solid supports of material such as glass or SiO.sub.2. 
"DNA chips" produced in this manner are useful for detecting or capturing 
multiple nucleic acid targets, for determining the nucleic type sequence 
of a target nucleic acid, for simultaneous analysis of the expression of 
thousands of genes, large scale gene discovery, DNA polymorphism 
screening, and mapping of genomic DNA clones, and are well suited for use 
in medical diagnostic assays for detection of pathogen infection and 
genetic mutation (for example, see U.S. Pat. No. 5,445,934; U.S. Pat. No. 
5,503,980; U.S. Pat. No. 5,605,662; Caviani-Pease et al., 1994, PNAS, Vol. 
91, pages 5022-6; and reviews by Ramsay, 1998, Nature Biotechnology, Vol. 
16, pages 40-44; and Marshall et al., 1998, Nature Biotechnology, Vol. 16, 
pages 27-31). 
Fodor et al. (U.S. Pat. No. 5,445,934, col. 3-21, 23-32) describes 
photolithographic solid-phase synthesis of arrays of oligomers, including 
arrays of oligonucleotides of known nucleotide sequence. The oligomer 
arrays are synthesized on a substrate by attaching photo-removable groups 
to the surface of a substrate, exposing selected regions of the substrate 
to light to activate those regions, and attaching monomeric subunits with 
photo-removable groups to the activated regions. The steps of 
photo-activation and attachment can be repeated until oligomers of desired 
length and sequence are synthesized. According to the current state of the 
art pertaining to the photolithographic synthesis of polynucleotide 
arrays, there is only a 92-94% chance that a new nucleotide will be 
incorporated where desired (McGall et al., J. Am. Chem. Soc., 1997, vol. 
119, pages 5081-90). Current technology thus imposes certain constraints 
on the possible array configuration, such as a practical upper limit on 
the number of nucleotides of approximately ten. 
McGall et al. (U.S. Pat. No. 5,412,087, col. 4-20) describes substrates 
with surfaces to which are attached compounds having a thiol functional 
group protected by a photo-removable protecting group, which compounds can 
be used to construct arrays of immobilized anti-ligands, such as 
oligonucleotide probes. 
Heller et al. describe a "master" DNA chip comprising a controllable, 
integrated array of micro-electrodes, and teaches denaturing 
double-stranded complexes comprising oligonucleotides at selected sites by 
increasing the negative potential and by use of chemical denaturants, in a 
process in which the oligomers hybridized at the selected sites are 
transferred to, or "printed" onto, another chip (U.S. Pat. No. 5,605,662, 
col. 20, lines 16-39). 
DNA oligonucleotides of defined sequence can also be used as structural 
components of an electronic computer chip (Hollenberg et al., U.S. Pat. 
No. 5,561,071). 
As is apparent from the preceding discussion there are numerous 
computational, data transmission-related, molecular biological, 
biochemical, and diagnostic applications which require the use of sets of 
oligonucleotides or oligonucleotide analogs of defined sequence and 
length. There currently is a need for a method for rapidly and efficiently 
providing the various combinations of oligomers required for applications 
such as those discussed above. 
BRIEF SUMMARY OF THE INVENTION 
Presented here is a rapid and efficient method for providing a selected set 
of oligonucleotides and/or oligonucleotide analogs comprising known 
subunit sequences. The method comprises the steps of 
a) obtaining a device for storing and providing oligomers comprising a 
substrate that supports an array of oligomer depots; 
wherein each depot comprises a surface to which are attached a plurality of 
oligonucleotides and/or oligonucleotide analogs having a selected subunit 
sequence; 
wherein the subunit sequence of the oligomers attached to at least one of 
said depots is different from the subunit sequence of the oligomers 
attached to a different depot of said array; and 
wherein oligonucleotides and/or oligonucleotide analogs comprising selected 
subunit sequences are stored at a plurality of depots of said array by 
being hybridized by Watson-Click pairing to the oligomers attached to the 
surfaces of said depots to form double-stranded complexes; 
b) locally denaturing double-stranded complexes of at least one selected 
depot of the intact array to release oligomers stored therein, without 
effecting significant denaturation of double-stranded complexes of the 
unselected depots of the array; and 
c) collecting the oligomers released as a result of locally denaturing 
double-stranded complexes of said at least one selected depot. 
The substrate that supports the array of oligomer-storing depots can be 
flexible, e.g., a nylon filter, or it can be of a rigid material such as 
SiO.sub.2 in a DNA chip. 
The array of depot sites may consist of from 2 to 10.sup.7 delimited areas 
wherein as many different types of oligomers are stored. The diameter of 
the area of each oligomer depot surface to which oligomers are attached 
can range from about 1 micron to 1 centimeter or more. Using known methods 
and currently available technology, one skilled in the art can readily 
fabricate an array of depot sites which are 5-10 microns in diameter, in 
which array the array density is about 10.sup.6 depot sites per cm.sup.2. 
Oligomers comprising a selected subunit sequence can be attached at a depot 
site directly to the area of substrate surface delimited by the depot 
boundaries, or they may be attached to the surface of a separate layer of 
material that is, in turn, attached to the substrate surface at the depot 
site. 
Oligomers are attached to their respective depot sites using protocols 
known by those skilled in the art for attaching oligomers to a substrate 
so that the attached oligomers are able to hybridize efficiently with 
nucleic acids comprising a complementary nucleotide sequence. 
The oligomers of known sequence attached to the array of depot sites can be 
synthesized by methods for synthesizing oligonucleotides and 
oligonucleotide analogs which are known to those skilled in the art. For 
example, they can be synthesized in situ on the supporting substrate, e.g. 
by photolithographic methods, or they can be pre-synthesized and deposited 
at the depot site, e.g. by micropipette, for chemical attachment. 
The present invention also features a method wherein the oligomers of known 
subunit sequence that are attached to the array of depot sites are 
synthesized by a novel method which uses local melting of hybridized DNA, 
DNA ligase, and a restriction enzyme. 
In all of the procedures involved in storing and releasing selected 
oligomers according to the present invention, the depot surfaces to which 
oligomers are attached are immersed in, or in contact with, buffered 
solutions of composition suitable for the biochemical or molecular 
biological operations being carried out. 
Depot sites within the array are thermally insulated and/or physically 
separated from each other so that denaturation of double-stranded oligomer 
complexes at the selected depots does not cause denaturation of 
double-stranded complexes at the non-selected depots. 
A collection of soluble oligomers of known composition is obtained by 
locally denaturing double-stranded complexes of the depots of the intact 
array comprising the desired oligomers, to yield the desired 
single-stranded oligomers in quantity related to the time and extent of 
the denaturing treatment. The oligomers are then collected in the buffer 
solution in which the array is immersed, for use in whatever application 
is contemplated. 
A storage device comprising 10.sup.6 storage depot sites is able to store 
every possible oligomer 10-mer sequence (4.sup.10 is approximately equal 
to 1.0.times.10.sup.6). Using a storage device comprising about 10.sup.6 
depot sites and storing every possible 10-mer, it is possible, with the 
present invention, to rapidly provide primers or hybridization probes that 
are complimentary to sites in any target nucleic acid. Also using such a 
storage device according to the present invention, a primer or 
hybridization probe of length greater than 10 subunits can be obtained 
rapidly by providing a set of oligomers of selected subunit sequence 
which, when ligated end-to-end, produce the desired longer polynucleotide. 
Suitable applications for which oligomers can be provided according to the 
present invention include, but are not limited to, nucleic acid 
computation, nucleic acid amplification, polynucleotide synthesis by 
primer extension or by ligating oligonucleotides together end-to end, 
nucleic acid hybridization for detection or isolation of a target nucleic 
acid, and data encryption and transmission. 
The present invention offers the advantages of rapidly and efficiently 
providing diverse, custom sets of oligomers, as needed, from a compact and 
easily accessed storage device. The invention is particularly advantageous 
for nucleic acid computation, and for encryption and transmission of data 
in the form of selected sets of hybridizing oligomers, since numerous sets 
of different oligomers having particular length and sequence properties 
are needed for both of these technologies, and these can be provided with 
facility by the present invention.

DETAILED DESCRIPTION OF THE INVENTION 
This invention features methods wherein custom sets of oligonucleotides 
and/or oligonucleotide analogs having selected subunit sequences are 
rapidly and efficiently provided by their controlled release from depot 
sites of an oligomer storage device. 
The practice of the present invention will employ, unless otherwise 
indicated, conventional techniques of chemistry, biochemistry, molecular 
biology, recombinant DNA, and medical diagnostic technology, which are 
within the capabilities of a person of ordinary skill in the art. Such 
techniques are explained in the literature. See, for example, Sambrook et 
al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 
1-3, Cold Spring Harbor Laboratory Press; B. Roe, J. Crabtree, and A. 
Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley 
& Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: 
Principles and Practice; Oxford University Press; M. J. Gait (Editor), 
1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and, D. 
M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA 
Structure Part A: Synthesis and Physical Analysis of DNA Methods in 
Enzymology, Academic Press. Each of these general texts are herein 
incorporated by reference. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are described. 
Nucleic Acid Oligomers 
The brief summary of DNA and RNA which follows is not meant to be 
exhaustive of the subject, but merely to provide a general framework for 
understanding the present invention. A more complete description of DNA 
and RNA technology is available in a number of texts, including: J. D. 
Wilson, M. Gilman, J. Witkowski, and M. Zoller, 1992, "Recombinant DNA", 
Second Edition, Scientific American Books; and, B. Lewin, 1997, "Genes 
VI", Oxford University Press. Each of these general texts are herein 
incorporated by reference. 
As set forth above, the present invention relates to nucleic acid 
biochemistry and molecular biology. Genetic information is stored, 
transmitted, and expressed by nucleic acids, DNA and RNA, which are 
constructed of nucleotide subunits. In general, oligonucleotides are 
linear sequences of a few nucleotides (the Greek-derived prefix 
oligo-indicates "a few"), while linear sequences of many nucleotides are 
called polynucleotides (the Greek-derived prefix poly- indicates "many"). 
The choice of whether to refer to a nucleic acid of a given number of 
nucleotide subunits as a polynucleotide or as an oligonucleotide is 
arbitrary. Oligomers are linear sequences of relatively few subunits. A 
number followed by the suffix -mer refers to an oligomer of the indicated 
number of nucleotide subunits. For example, an oligomer that contains 12 
or 17 bases is referred to as a 12-mer or as a 17-mer, respectively. Each 
nucleotide contains a phosphate group, a sugar moiety, and either a purine 
or pyrimidine base. The sugar of DNA is deoxyribose while the sugar of RNA 
is ribose. Nucleosides consist of a purine or pyrimidine base attached to 
ribose or deoxyribose. Polynucleotides and oligonucleotides each consist 
of a linear sequence of nucleotides of DNA or RNA in which the 3' position 
of the sugar of one nucleotide is linked through a phosphate group to the 
5' position of the sugar on the adjacent nucleotide. Ligation is the 
formation of the phosphodiester bond which joins the adjacent nucleotides 
in the same nucleic acid chain. Two purine bases and two pyrimidine bases 
are found in both DNA and RNA. The purines adenine (A) and guanine (G) and 
the pyrimidine cytosine (C) occur in both DNA and RNA. However, thymine 
(T) only occurs in DNA and uracil (U) only occurs in RNA. The nucleotides 
of DNA are deoxyadenylic acid, thymidylic acid, deoxyguanilic acid, and 
deoxycytidylic acid, while the corresponding nucleotides of RNA are 
adenylic acid, uridylic acid, guanylic acid, and cytidylic acid. The 
sugar-phosphate backbones are on the outside of the DNA molecule and the 
purine and pyrimidine bases are on the inside, oriented in such a way that 
they can form hydrogen bonds to bases on opposing chains. Adenine (A) can 
pair only with thymine (T), while guanine (G) can bond only with cytosine 
(C). Hybridization is the process by which two complementary RNA and DNA 
strands pair to produce an RNA-DNA hybrid, or by which two complementary 
DNA single strands pair to produce a DNA--DNA hybrid, also known as 
double-stranded DNA. Universal base analogues or universal nucleotides are 
capable of hybridizing with any one of the four DNA nucleotides (Nichols 
et al., Nature, 1994, Vol. 369, pages 492-3; and Loakes et al., Nucleic 
Acids Research, 1994, Vol. 22, pages 4039-43). An example of a universal 
base analogue is 5-Nitroindole (Loakes et al., Nucleic Acids Research, 
1994, vol. 22, pages 4039-43). 
As used herein, the term oligomers refers to RNA or DNA oligonucleotides, 
RNA or DNA oligonucleotide analogs, or a combination of RNA and/or DNA 
oligonucleotides and RNA and/or DNA oligonucleotide analogs, which can be 
attached to the storage device depot sites, or which can be stored by 
being hybridized to oligomers attached to the depot sites. 
Depending on the purposes for which the oligomers are to be used, the RNA 
or DNA oligonucleotide analogs can be oligomers in which from one to all 
nucleotide subunits are replaced with a nucleotide analog to confer 
desired properties such as detectability, increased hybridization 
affinity, resistance to degradation by nucleases, or the ability to 
covalently modify a target nucleic acid. Such oligonucleotide analogs 
include but are not limited to oligomers comprising 2'-O-alkyl 
ribonucleotides, phosphorothioate or methylphosphonate internucleotide 
linkages, peptide nucleic acid subunits (see U.S. Pat. No. 5,714,331, in 
entirety), and nucleotides modified by attachment of radioactive, or 
fluorescent groups, groups which intercalate, cross-link or cleave a 
nucleic acid, or groups which alter the electronegativity or 
hydrophobicity of the oligomers. Methods for making and using 
oligonucleotides and oligonucleotide analogs such as those listed above 
are well known to those skilled in the art of making and using 
sequence-specific hybridizing oligomers. 
The sizes of the oligomers attached to the depot site surfaces, and of the 
oligomers stored at the depots, can range from about 4 subunits to 1000 or 
more subunits in length. The stored oligomers can be longer, shorter, or 
the same length as the attached oligomers. Oligomers having different 
lengths, and oligonucleotide analogs having different chemical structures 
and properties, can be stored in different depots of the same array. Those 
skilled in the art appreciate that oligomer hybridization specificity and 
affinity are determined, in part, by the length and chemical structure of 
the oligomer, and are able to select the structural parameters of the 
oligomers attached to, and stored in, the depots of the oligomer-storing 
device that are appropriate for their intended use. For example, the 
subunit sequences of the attached and stored oligomers can be selected so 
that they do not comprise self-complementary sequences that stabilize 
folding of said oligomers into hairpin structures which interfere with 
formation of inter-strand duplexes. Additionally, the subunit sequences of 
the attached and stored oligomers can be selected so that the melting 
temperatures (Tm) of the double-stranded complexes formed by hybridization 
of the complementary portions of the attached and stored oligomers at all 
of the depots of the array are within a selected range, e.g., in the range 
of a selected Tm plus or minus about 5 degrees C., for more efficient 
control of oligomer storage and release. 
The Oligomer Storage Device 
A central feature of the present invention is that the desired set of 
oligomers is provided from an oligomer storage device comprising a 
substrate (for example, see (1) in FIGS. 1 and 2) supporting an array of 
oligomer-storing sites, referred to herein as depots. The substrate can 
have a flat surface that supports the array, or it can be distributed in 
three dimensions, such as in a gel, a fibrous or granular matrix, or in a 
porous solid. By depot is meant a site at which oligomers are stored 
comprising a delimited area or volume that is part of or attached to the 
supporting substrate, to which are attached hybridizing oligomers 
comprising a selected subunit sequence (for example, see (2) in FIGS. 1 
and 2). A depot site can have any size, shape, or volume, consistent with 
the objective of the invention of storing and selectively releasing 
oligomers as needed. By array is meant an arrangement of locations in or 
on the oligomer-storing device. The locations can be arranged in 2- or 3- 
dimensional arrays, or other matrix formats. FIG. 1 shows a 2-dimensional 
4.times.5 array of depots on a supporting substrate. The number of 
locations in the array can range from 2 to 10.sup.7 or more. It is within 
the knowledge of those skilled in the art to fabricate a rigid substrate 
supporting an array of oligomer depot sites that can range in diameter 
from about 1 micron to 1 centimeter or more (see U.S. Pat. No. 5,412,087, 
col. 8, lines 50-68; U.S. Pat. No. 5,445,934, col. 9, lines 10-18; and 
Ramsay, Nature Biotechnology, vol. 16, p. 40, 1998). All of the depot 
sites of a given array can have the same diameter, or a single depot array 
can comprise depot sites having different diameters. The preferred method 
of the present invention features storing about 10.sup.2 to 10.sup.7 
different types of oligomers of about 8 to 30 subunits in length in a 
micro-array of thermally isolated depot sites on a rigid substrate. 
A substrate which is suitable for supporting immobilized nucleic acids for 
hybridization analysis can, in general, be adapted for use as an oligomer 
storage device of the present invention. Accordingly, a variety of 
different designs and materials are available for preparing the oligomer 
storage device of the present invention. For example, the storage device 
may be a flexible filter, e.g., of nylon or nitrocellulose, or it may be 
of a rigid material such as silica, silicon, glass, crystalline Al.sub.2 
O.sub.3 ("synthetic sapphire"), beryllium oxide, or a solid substrate 
coated with a noble metal such as gold. Methods for making such substrate 
supports for hybridizing oligomers are well known to those skilled in the 
art. (See U.S. Pat. No. 5,412,087, col. 6, lines 1-39; U.S. Pat. No. 
5,445,934, col. 11, lines 49-63; Ramsay, Nature Biotechnology, vol. 16, 
pages 40-41; Drmanac et al., Genomics, 1989, vol. 4, pages 114-128; Mirkin 
et al., Nature, vol. 382, pages 607-609, 1996; R. Corn, DNA Computing 
Overview, last modified Mar. 13, 1998, &lt;http://www. 
corninfo.chem.wisc.edu/writings/DNA overview.html&gt;). 
The oligomers attached at the depot sites can be attached directly to the 
surface of the substrate, or to the surface of a pad or pedestal-like 
structure that is in itself attached to the substrate, which pad or 
pedestal-like structure can be of material that is the same or different 
from that of the substrate. FIG. 2 shows oligomers attached to a depot 
site (2) comprising a pad comprising three different layers ((10), (11), 
and (12)) affixed to a rigid transparent substrate (1). The depot surface 
to which the oligomers are attached can be located on a raised feature or 
in a well-like depression on the surface of the supporting substrate. 
Methods for making arrays comprising oligomers attached to depot sites to 
produce oligomer-storing devices for the present invention are well known. 
Such methods include in situ synthesis of oligomers attached at their 3' 
ends to a functionalized surface such glass, SiO.sub.2, or GaAs (for 
example, see U.S. Pat. No. 5,445,934, col. 23, line 3, to col. 25, line 
18; U.S. Pat. No. 5,412,087, col. 4, line 67 to col. 10, line 35; U.S. 
Pat. No. 5,605,662, col. 17, lines 21-63). Alternatively, pre-synthesized 
oligomers can be chemically attached to the substrate, e.g., by 
derivatizing the oligomers or the attachment surface, and then depositing 
microdroplets of the oligomers at the appropriate depot sites and allowing 
the oligomers to react with the depot site surface, or by attaching 
biotinylated oligomers to a streptavidin-coded surface (see U.S. Pat. No. 
5,503,980, col. 13, lines 2-9; U.S. Pat. No. 5,412,087, col. 1, line 18 to 
col. 3, line 13 and col. 6, line 21 to col. 10, line 35; Marshall et al., 
Nature Biotechnology, vol. 16, pages 27-29, 1998; and Mirkin et al., 
Nature, vol. 382, pages 607-609, 1996). 
A preferred mode of attachment of oligomers to depot surfaces for use 
according to the present invention is to use uncharged spacer groups ((6) 
in FIG. 2) to tether the oligomers to the depot surface (U.S. Pat. No. 
5,445,934, col. 11, line 49, to col. 13, line 45; Caviani-Pease et al., 
P.N.A.S., 1994, vol. 91, pages 5022-24), as the use of such spacer groups 
is known to increase hybridization efficiency (Marshall et al., Nature 
Biotechnology, 1998, vol. 16, page 29). 
Enzymatic Synthesis of Oligomers in situ 
An additional and novel method for making a substrate-supported array of 
oligomer depot sites which can be used as an oligomer-storing device for 
the present invention is described as follows. A double-stranded DNA 
consisting of an .alpha. strand and a complementary .beta. strand, denoted 
.alpha.-.beta. (alpha-beta), is synthesized by a known method of 
oligonucleotide synthesis (see M. J. Gait (Editor), 1984, Oligonucleotide 
Synthesis: A Practical Approach, IRL Press). One to four or more unpaired 
nucleotides at the phosphorylated 5' end of the .alpha. strand extend 
beyond the 3'-hydroxyl-terminated end of the complementary .beta. strand 
as a single-stranded structure that is referred to as a "sticky end," 
because it can hybridize to another single-stranded nucleic acid having a 
complementary nucleotide sequence. The sticky 5' ends of the .alpha. 
strands are the sites where new nucleotides are added to the desired 
oligonucleotides being synthesized. Alternatively, the orientation of the 
strands of the duplex .alpha.-.beta. oligomer with respect to the sticky 
end may be reversed, although suitable restriction enzymes needed in the 
nucleotide addition step discussed below are more rare in this case. New 
nucleotides may be added to the duplex .alpha.-.beta. oligomers in a 
reaction in which the .alpha.-.beta. oligomers are free in solution, or 
are attached to a substrate, as shown in FIGS. 3 and 4. 
In one embodiment, a substrate is uniformly covered with duplex 
.alpha.-.beta. oligomers, the DNA-covered surface is divided into local 
regions referred to as depots, and a different oligonucleotide sequence is 
synthesized in each depot. The duplex .alpha.-.beta. DNA molecules are 
synthesized and attached to the substrate using known protocols; for 
example, .alpha. oligomers can be synthesized in situ on the substrate by 
a photolithographic method, and .beta. oligomers can be synthesized by 
routine chemical methods and hybridized to the attached .alpha. oligomers; 
pre-fabricated .alpha.-.beta. DNA molecules can be covalently attached to 
functionalized substrate SiO groups, biotinylated DNA oligomers can be 
bound to a streptavidin-coated surface, or thiolated DNA oligomers can be 
linked to a gold substrate, as discussed above. It is preferred that the 
3' end of the .alpha. strand of the duplex .alpha.-.beta. DNA oligomer be 
anchored to the substrate through an uncharged spacer group; however, the 
orientation of the strands of the duplex .alpha.-.beta. oligomer with 
respect to the substrate may be reversed, although suitable restriction 
enzymes needed in step 6 below are more rare in this case, as noted above. 
Synthesis of a different oligonucleotide sequence in each depot is achieved 
by a sequential series of hybridization, ligation, melting, and cleaving 
reaction, in which each depot is locally heated in turn so that .epsilon. 
(epsilon) DNA strands comprising the new nucleotides to be added hybridize 
only to DNA strands of the depot where addition is to occur. Localized 
heating of the DNA oligomers of the claimed invention may be achieved by 
any suitable means in accord with the types of oligonucleotides being 
synthesized, the type of substrate used, and the embodiment of the 
invention being employed. Suitable methods for locally heating depot sites 
are discussed in detail below. The temperature for heating is selected, 
with consideration to the lengths and sequences of the oligomers and to 
the ionic strength of the reaction solution, to rapidly melt off undesired 
DNA strands bound to the .gamma. strands without melting the 
.alpha.-.beta. duplex structures, so that the desired .epsilon. strands 
with the nucleotides to be added can hybridize to the exposed .gamma. 
strands. 
The synthesis of DNA strands according to the invention is illustrated as 
follows, referring to FIGS. 3 and 4 in embodiments in which the duplex 
.alpha.-.beta. oligomers are attached to and uniformly cover a substrate. 
One possible substrate is comprised of a wafer of Si covered by (1) a 
thermally-insulating 1 .mu.m thick layer of SiO.sub.2, (2) a heat 
absorbing 0.5 .mu.m thick layer of amorphous Si and (3) a 0.5 .mu.m thick 
layer of SiO.sub.2 upon which to anchor the DNA oligomers (see elements 
(10), (11), and (12), respectively, in FIG. 2). The substrate may be 
patterned into 10 .mu.m.times.10 .mu.m pads to better define and thermally 
isolate the identifiable areas (depots) of the plate. The .alpha. strand 
of the duplex .alpha.-.beta. DNA oligomer is anchored to the substrate at 
its 3' end, and one to four or more unpaired nucleotides at its 
phosphorylated 5' end extend beyond the 3'-hydroxyl-terminated end of the 
complementary .beta. strand to form a sticky end. In a preferred 
embodiment, the Si substrate is replaced with a substrate of transparent 
crystalline Al.sub.2 O.sub.3 to allow back illumination of the desired 
depots, thus protecting the DNA from direct exposure to the laser 
radiation. Steps of hybridization, ligation, heating to melt desired 
portions of the duplex DNA complexes, and cleavage by restriction enzyme, 
are carried out in suitable buffered solutions for these reactions which 
are well known to those skilled in the art (see Sambrook et al. and the 
other previously cited references teaching biochemical and molecular 
biological methodology). In embodiments in which DNA molecules are 
synthesized on a substrate, the DNA-covered substrate is immersed in 
suitable buffer during each reaction step of the method. 
Step (1): A set of single-stranded .gamma. (gamma) DNA oligomers is 
prepared having phosphorylated 5' ends, and in which the nucleotide 
sequences at the 5' ends are randomly varied so that individual members of 
the set of .gamma. strands can hybridize with every possible a strand 
sticky end. The set of .gamma. oligomers is allowed to hybridize with the 
5' sticky ends of the .alpha. strands. The bases of a number p of 
nucleotides in each .gamma. strand adjacent to the randomized 5'-terminal 
nucleotides are universal bases, where p is the number of new nucleotides 
to be transferred from the .epsilon. strands to the ends of the .alpha. 
strands. In theory, p can range in value from 1 to as large a number as 
desired. In using the invention to make a set of long oligonucleotides 
which differ from each other at only one or a few nucleotides, it may be 
practical to use oligomers having large p, so as to add large blocks of 
nucleotides to the ends of the .alpha. strands in a single step. In using 
the invention to make an array of highly variable oligonucleotides, the 
upper value of p is limited by the practical need to repeat the steps for 
adding p nucleotides up to 4.sup.p different times for each set of p 
nucleotides that are added. 
Step (2): In the presence of T4 DNA ligase and ATP, the 5' ends of the 
.gamma. strands become ligated to the 3' ends of the .beta. strands. 
Step (3): Desired strands .epsilon. (epsilon) are introduced to hybridize 
to the .gamma. strands, wherein the .epsilon. strands have p nucleotides 
at their 3' ends which are to be added to the .alpha. strands. Since there 
are 4.sup.p different types of .epsilon. strands, p being the number of 
bases added in each step, with each different .epsilon. strand ending in 
one of the 4.sup.p possible sets of p bases, this step would need to be 
repeated once for each of the different p-tuples of bases added to the 
entire substrate or plate, prior to ligating. The .epsilon. strands 
hybridize with the .gamma. strands, with the p bases to be added pairing 
with the p universal bases on the .gamma. strands. The overall lengths and 
nucleotide sequences of the .gamma. and .epsilon. oligomers are selected 
so that .gamma. and unligated .epsilon. strands form a duplex structure 
that melts at a temperature at which the .alpha.-.beta. duplexes remain 
intact. A preferred configuration for the .alpha.-.beta.-.gamma.-.epsilon. 
complex is one wherein the 5' ends of .epsilon. strands and 3' ends of 
.gamma. strands form blunt ends. 
Step (4): In the case where a single type of oligonucleotide is being made, 
nicks between the 3' hydroxyl terminations of the .epsilon. strands and 
the 5' phosphate terminations of the .alpha. strands are ligated according 
to Step 5 below. 
In the case where an array of different substrate-bound oligonucleotides is 
being synthesized, with new nucleotides also being added to the ends of 
DNA oligonucleotides at other locations on the substrate, the undesired 
.epsilon. strands are removed by local heating without melting the 
.alpha.-.beta. duplex portions, for example, by using laser illumination 
patterned with a lithographic mask, and are washed away. Desired .epsilon. 
strands are then hybridized to exposed .gamma. strands of substrate-bound 
DNA molecules at the heated locations, by repeating Step 3. Steps 4 and 3 
of heating to selectively remove undesired .epsilon. strands, and then 
hybridizing desired .epsilon. strands at each location where nucleotides 
are to be added, are repeated until all locations where nucleotides to be 
added to the sticky ends of the substrate-bound DNA have been treated. 
Step (5): After all desired .epsilon. strands are hybridized to the growing 
DNA molecules, nicks between the 3' hydroxyl terminations of the .epsilon. 
strands and the 5' phosphate terminations of the .alpha. strands are 
ligated using T4 DNA ligase again. 
Step (6): The resulting double-stranded DNA molecules are cut with a 
restriction enzyme that leaves a new sticky end similar to the original 
.alpha.-.beta. sticky end, except that cleavage results in addition of p 
new nucleotides to the 5' end of the .alpha. strand. Cleavage may also 
result in addition of one or more paired nucleotides to the 3' end of the 
.beta. strand. In the preferred method, the restriction enzyme that is 
used is one that cuts at a site adjacent to, but outside of, its specific 
recognition sequence that is built into the .epsilon.-.gamma. sequence, to 
leave the new sticky end on the growing double-stranded oligonucleotide. 
An example of such a restriction enzyme which is suitable for use in the 
invention is Alw 26 I. Restriction enzyme recognition sites in the growing 
.alpha.-.beta. duplex can be protected from unwanted cleavage by 
methylation of one or both strands at the enzyme recognition site in the 
.alpha.-.beta. duplex to be protected, using the appropriate methylase 
enzymes, or by incorporation of a methylated nucleotide or a 
restriction-enzyme-inhibiting nucleotide analog, which incorporation could 
be carried out during synthesis of the original .alpha.-.beta. duplex 
stem, or in the step wherein new bases are added to the growing duplex DNA 
molecule. 
Step (7): The process is repeated for each new set of bases to be added to 
the growing duplex DNA molecules. 
One skilled in the art can readily design the original .alpha. and .beta. 
oligomers to comprise a recognition site for a restriction enzyme that is 
different from the one used in the synthetic reactions, so that the 
polymers can be released after synthesis, if desired. 
An alternate and less-preferred procedure is illustrated in FIG. 4 in which 
steps 3-5 are modified to include use of protective .delta. strands as 
follows: 
Step (3, modified): An excess of .delta. (delta) protector strands are 
prepared which are perfectly complementary to all of the nucleotides of 
the single-stranded portion of the .gamma. strands extending from the 
.alpha.-.beta. duplex, except that the .delta. strands comprise 
3'-phosphate-terminated ends, or they lack a complementary nucleotide at 
their 3' ends, so that unwanted ligation of the 3' ends of the .delta. 
strands to the 5'-ends of the .alpha. strands is prevented. The excess of 
.delta. protector strands are introduced to hybridize to and protect the 
.gamma. strands in non-reacting depots from binding to nucleotide-adding 
.epsilon. strands. 
Step (4, modified): In desired locations, the protector .delta. strands are 
melted off the .gamma. strands by local heating, for example, by using 
laser illumination patterned with a lithographic mask, and are washed 
away. Desired strands .epsilon. (epsilon) are then introduced to hybridize 
to the single-stranded .gamma. oligomers. The remaining steps of the 
alternate method are as described above for the method in which protector 
.delta. strands are not used. If one wants to make some of the strands 
shorter than normal, so that the .delta. strands need to be left in place 
during the restriction step, the strands containing a .delta. may be 
protected from cutting by methylation of the restriction enzyme 
recognition site on the .delta. strand. 
The fidelity of synthesis attained using the above-described method for 
oligomer synthesis of the present invention permits efficient and accurate 
synthesis of oligonucleotides in substrate-bound arrays that are 
considerably longer than those that can be accurately made using current 
technologies; for example, substrate-bound oligonucleotides of up to 20, 
30, 50, or even 100 or more nucleotide subunits, can be accurately made by 
the present invention. 
Storing Oligomers in Depots 
Oligomers are stored in the depot array of the storage device by allowing 
them to hybridize specifically to oligomers comprising complementary 
subunit sequences which are attached at the depot sites ((2) in FIG. 2), 
to form double-stranded oligomer complexes attached to the depot sites 
((7) in FIG. 2). Those skilled in the art recognize that the number of 
consecutive complementary nucleotides that must be present in an 
oligonucleotide so that it hybridizes specifically to a target nucleic 
acid molecule can vary considerably, from about 4 up to 14 or more, 
depending on such factors as the complexity of the set of target nucleic 
acids and the physical conditions (ionic strength, temperature, anionic 
and cationic reagents, etc.) used in the hybridization and wash steps. The 
statement that a soluble oligomer hybridizes specifically to a 
substrate-bound oligomer or other target nucleic acid is intended to mean 
that a portion of the oligomer comprising a nucleotide sequence 
complementary to a sequence in the substrate-bound oligomer or other 
target nucleic acid binds by Watson-Crick base-pairing to the 
complementary portion of the substrate-bound oligomer or other target 
nucleic acid to form a stable double-stranded complex, under hybridization 
conditions that are sufficiently stringent that oligomer molecules having 
fewer bases complementary to, or forming less stable duplex structures 
with, said substrate-bound oligomers or other target nucleic acids do not 
hybridize to said substrate-bound oligomers or other target nucleic acids 
and form stable double-stranded complexes. Selection of parameters such as 
the lengths of the complementary portions of the soluble and 
substrate-bound oligomers and the conditions used in hybridization and 
wash steps, so that the soluble oligomers hybridize specifically to their 
substrate-bound counterparts, is well within the capabilities of a person 
of ordinary skill in the art (e.g., see Sambrook et al., 1989, supra, 
Chapter 11). 
For example, a complete set of oligonucleotides comprising every possible 
sequence of n consecutive nucleotide subunits can be stored in an array of 
4.sup.n depot sites comprising complementary oligomers by exposing the 
array to the soluble oligomers at a temperature about 25.degree. C. below 
the lowest melting temperature for the set of double-stranded complexes to 
be formed, in a suitable buffer containing a high molar concentration of 
Na.sup.+. The time required to saturate the 4.sup.n depot sizes with the 
n-mer oligomers is known to be dependent on the concentrations of the 
oligomers, the temperature, and the concentration of Na.sup.t ions. If the 
soluble oligonucleotides are applied at a concentration of 0.5 mole of 
single nucleotides per liter and the Na.sup.+ concentration is 1 mole per 
liter, the time for half of the hybridization reaction to be completed is 
about 4 seconds for n=10, and about 100 days for n=20. (Britten et al., 
Methods in Enzymology, 1974, vol. 29, part E, pages 363-418; Wetmur et 
al., J. Molecular Biology, 1989, vol. 31, page 349; Britten et al., 
Science, 1968, vol. 161, page 529). 
Releasing Selected Oligomers 
A custom set of soluble oligomers of known composition is obtained by 
locally denaturing double-stranded complexes of selected depots of the 
intact array comprising the desired oligomers, and collecting the 
oligomers released from the selected depots ((5) in FIG. 2) into the 
buffer solution in which the array is immersed ((8) in FIG. 2). 
Denaturation of oligomer complexes at selected depots can be achieved by 
any of the nucleic acid-denaturing treatments known to those skilled in 
the art of nucleic acid biochemistry. Those skilled in the art appreciate 
that the melting temperature of a double-stranded oligonucleotide complex 
is dependent on the length, nucleotide sequence, and chemical structure of 
the complex, and on the ionic strength and chemical composition of the 
solvent (see Sambrook et al., 1989, supra, page 11.46). 
The preferred method for denaturing double-stranded complexes at the 
selected depots to release the desired oligomers is by locally heating the 
selected depots so as to subject the selected depots to a raised 
temperature under appropriate solution conditions for a period of time 
sufficient to release the desired oligomers from the selected depots. 
Localized heating of the selected depot surfaces can be achieved by any 
suitable means in accord with the structure and size of the supporting 
substrate, and the size and disposition of the individual depot sites. For 
example, selected depots can be locally heated by illuminating the surface 
of the array, in a suitable buffer and at a temperature below the melting 
point of the oligomer duplexes, with a pattern of focused irradiation from 
a radiant energy source ((4) and (9) in FIG. 2), e.g. an argon laser, that 
heats only those depots storing the desired oligomers. The laser can be 
mounted on a support which provides precise x-y translation control, to 
permit controlled heating of one depot at a time, in serial fashion. 
Alternatively, the laser can have a broad beam that can irradiate a mask, 
the image of which can irradiate all of the depots in the array at once. 
The mask can thus be used to shield the unselected depots so that only 
those comprising the desired oligomers are heated. To heat a single depot 
having a surface area of about 100 .mu.m.sup.2 to about 70.degree. C. in a 
suitable buffered solution to locally melt double-stranded DNA duplexes 
stored at the heated depot will require roughly 10 milliwatts of argon 
laser light (488 nm). Use of a substrate which is transparent to argon 
laser light, e.g. crystalline Al.sub.2 O.sub.3, to support thermally 
isolated, light-absorbing, depot surfaces to which the oligomers are 
attached, allows back illumination of the desired depots as shown in FIG. 
2, thus protecting the oligomers from direct exposure to the laser 
radiation. A substrate of Al.sub.2 O.sub.3 is also advantageous because 
the high thermal conductivity of Al.sub.2 O.sub.3 permits the substrate to 
act efficiently as a heat sink, by drawing heat away from the irradiated 
depot sites and so providing greater thermal isolation of the unselected 
depot sites. Alternatively, the storage device substrate comprising the 
depot array could be in contact with, or have integrated within it, a 
controllable, addressable, array of resistive heating elements which is 
spatially aligned with the depot array, so that application of current to 
selected resistive heating elements locally heats selected depots proximal 
to the activated heating elements to release the desired oligomers. Heller 
et al. teach fabrication of a silicon substrate into which is integrated a 
micro-array of electronically addressable micro-locations corresponding to 
a micro-array of DNA storage sites (U.S. Pat. No. 5,605,662, col. 9-10, 
12-16). Accordingly, it is within the knowledge of those skilled in the 
art of microlithography and thick film circuitry to fabricate a DNA chip 
in which there is integrated an array of electronically addressable 
micro-locations comprising resistive heating elements such as can be 
formed, for example, by depositing undoped polycrystalline silicon at 
positions between addressable conducting wire grids (Kamins, 
Polycrystalline Silicon for Integrated Circuit Applications, 1988, Kluwer 
Academic Publications, Boston). As described by Heller et al., metal 
contact pads along the outside perimeter of the chip permit wiring such a 
chip comprising an integrated electronically addressable micro-array to a 
microprocessor-controlled power supply and interface for controlling the 
device (U.S. Pat. No. 5,605,662, col. 12). The amounts of oligomers 
released by localized heating can be controlled by varying the amount of 
heat applied, e.g., by controlling the intensity of the laser light or the 
temperature of the resistive heater, and/or by varying the time period 
during which heat is applied. According to the preferred method, the 
localized heating of selected depots to release desired oligomers stored 
therein is electrically controlled by a programmable microprocessor and an 
interface for controlling the process. By the method of the present 
invention, local heating of selected depots will cause oligomer duplexes 
at the heated depots to melt in a short time of the order of seconds, to 
yield single-stranded oligomers in quantities related to the time and 
extent of heating. 
Heller et al. teach that denaturation of DNA at selected depots can also be 
induced by locally increasing the negative electric potential at the 
selected depots (Heller et al., U.S. Pat. 5,605,662, column 20). Thus, an 
array of micro-electrodes integrated within, or closely associated with, a 
substrate supporting an oligomer-storing array of depot sites can be used 
to create denaturing conditions at selected depots of the array to 
practice the present invention. In addition positively charged chaotropic 
agents and other denaturants can be added to the solution in contact with 
the selected depots to promote denaturation of the attached 
double-stranded complexes. Exposure to denaturing solution conditions can 
be limited to the depots selected for denaturation by surrounding the 
selected depot surfaces with a liquid-impermeable barrier that prevents 
the denaturing solution from contacting non-selected depot surfaces. For 
example, individual depots of a large-scale array, in which depot surfaces 
are 0.1 to 10 mm or more in diameter, can be situated in wells or 
surrounded by raised divider walls to be "fluidically isolated" from each 
other, so that selected depot surfaces can be exposed to denaturing 
solution without also exposing non-selected depot surfaces to the 
denaturing conditions. Denaturation of selected depots, whether by 
localized heating, application of increased negative potential, denaturing 
solution, or any combination of these means, can be carried out serially, 
one depot at a time, or in parallel with multiple depots being treated 
simultaneously. 
Collecting and Using the Released Oligomers 
Oligomers released from selected depot sites following denaturation of 
double-stranded complexes at those sites ((5) in FIG. 2) are collecting by 
collecting the solution in contact with the treated depot surfaces ((8) in 
FIG. 2). The solution in contact with the oligomer-storing depot array can 
be enclosed or contained within a reservoir, and once the desired 
oligomers are released into the solution, it can be collected by any 
suitable means, e.g. by a manually operated or automated pipetting device, 
or a syringe. Alternatively, the solution containing the desired oligomers 
can be removed from the reservoir and transferred to a suitable collecting 
device, and fresh solution can be added to the reservoir in its place, 
e.g. to wash away residual oligomers in preparation for releasing a 
different set of oligomers, by using automated or 
microprocessor-controlled pumps which direct the flow of the different 
solutions through tubes connected to the reservoir. 
The collected oligomers may then be used in protocols which employ a 
customized set of oligonucleotides or oligonucleotide analogs. Such 
protocols include, but are not limited to, protocols for nucleic acid 
computation, nucleic acid amplification, polynucleotide synthesis by 
primer extension or by ligating together overlapping complimentary 
oligonucleotides, nucleic acid hybridization for detection or isolation of 
a target nucleic acid, and data encryption and transmission. 
EXAMPLES 
The following examples further demonstrate several preferred embodiments of 
this invention and are offered by way of illustration, but should not be 
construed as limiting the claims thereof. Those skilled in the art will 
recognize numerous equivalents to the specific embodiments described 
herein. Such equivalents are intended to be within the scope of the 
claims. 
Example 1 
Synthesis of a DNA oligonucleotide by the ligation/restriction method 
As a concrete example for the case in which a single base is to be added to 
the strands in each step (i.e., p=1), the following oligomers are selected 
to carry out the needed reactions: 
34-mer, .alpha.: 5' TCTTAACATAGGAATTTGAGGCAGTACGCAAAAA 3'-biotin (B) (SEQ 
ID NO: 1). 
30-mer, .beta.': 3' AGAATTGTATCCTTAAACTCCGTCATGCGT 5' (SEQ ID NO: 2). 
26-mer, .beta.: 3' TTGTATCCTTAAACTCCGTCATGCGT 5' (SEQ ID NO: 3). 
17-mer, .gamma.: 3' TCACGTCAGAGCNNNNN 5' (SEQ ID NO: 4), wherein the first 
N in the 3'.fwdarw.5' direction is a universal base and the subsequent N's 
designate A, C, G, or T. 
13-mer, .epsilon..sub.A : 5' AGTGCAGTCTCGA 3' (SEQ ID NO: 5). 
13-mer, .epsilon..sub.T : 5' AGTGCAGTCTCGT 3' (SEQ ID NO: 6). 
13-mer, .epsilon..sub.G : 5' AGTGCAGTCTCGG 3' (SEQ ID NO: 7). 
13-mer, .epsilon..sub.C : 5' AGTGCAGTCTCGC 3' (SEQ ID NO: 8). 
The sequence of oligomer SEQ ID NO: 2 (.beta.') consists of 18 A-T's and 12 
G-C's, chosen to minimize the number of A-T and/or G-C matches of the 
sequence with itself for shifts of up to .+-.20 bases. It is further 
chosen to have no more than 3 A's, T's, or G-C's in a row; no more than 2 
G's or 2 C's in a row. These selections are to ensure that the strands 
will not form hairpins. Oligomer SEQ ID NO: 2 (.beta.') is chosen to lack 
the restriction enzyme Alw 26 I recognition sequence GTCTC/CAGAG or either 
of the four base pair segments of that sequence. Oligomer SEQ ID NO: 1 
(.alpha.) is complementary to the full sequence of .beta.', and has in 
addition a quartet of A's and a biotin group at the 3' end for attaching 
the .alpha.'s to the substrate. SEQ ID NO: 3 (.beta.) is identical to 
.beta.' except that four bases are missing from the 3' end to produce a 
4-base sticky end when hybridized to .alpha.. In oligomer SEQ ID NO: 4 
(.gamma.), the first N in the 3'.fwdarw.5' direction is a universal base, 
such as 5-Nitroindole, and each of the subsequent N's are random 
deoxyribonucleotide bases. The concentration of any one particular version 
of .gamma. will be 1/256 of the total. The .epsilon. oligomers (SEQ ID 
NOs:5-8) each contain one of the two single-stranded sequences from the 
duplex DNA Alw 26 I restriction enzyme recognition sequence, which cuts 
leaving the 5' sticky end indicated: 
5' . . . NNNGTCTCN 3' (SEQ ID NO: 9, from the .epsilon. strand) 
3' . . . NNNCAGAGNNNNN 5' (SEQ ID NO: 10, from the .gamma. strand), 
wherein the 5.sup.th N from the 5' end of the .gamma. strand is a universal 
base, and the other Ns designate A, C, G, or T. 
The detailed steps in making a DNA hybridization array are as follows. 
Step (1). We start by attaching .alpha. oligomers uniformly over the 
substrate, e.g., by using the affinity of biotin for a streptavidin-coated 
glass surface, and .beta. strands are then hybridized with the anchored a 
strands, giving: 
5' TCTTAACATAGGAATTTGAGGCAGTACGCAAAAA 3'-B (.alpha., SEQ ID NO:1) 
3' TTGTATCCTTAAACTCCGTCATGCGT 5' (.beta., SEQ ID NO:3). 
Step (2). The set of .gamma. DNA strands (SEQ ID NO: 4) is introduced to 
hybridize with the sticky ends of the .alpha. strands, and the ends of the 
.gamma. DNA strands are ligated to the ends of the .beta. strands of the 
anchored .alpha.-.beta. DNA by incubating with T4 DNA ligase and ATP, 
giving: 
5' TCTTAACATAGGAATTTGAGGCAGTACGCAAAAA 3'-B (.alpha.) 
3' TCACGTCAGAGCNNNNNTTGTATCCTTAAACTCCGTCATGCGT 5' (.gamma.+.beta.) 
where the .alpha. strand is SEQ ID NO: 1, and the .gamma.+.beta. strand is 
SEQ ID NO: 11 wherein the first N in the 3'43 5' direction in .gamma. is a 
universal base and the subsequent N's designate A, C, G, or T. 
Step (3). The DNA-covered substrate is incubated in the presence of an 
oligomer denoted .epsilon..sub.X containing the base X to be added to the 
.alpha. strand, so that the .epsilon..sub.X oligomers hybridize to the 
.gamma. strands. In this example, .epsilon..sub.A =SEQ ID NO: 5; 
.epsilon..sub.T =SEQ ID NO: 6; .epsilon..sub.G =SEQ ID NO: 7; and 
.epsilon..sub.C =SEQ ID NO: 8. 
Step (4): To add one of the 4 bases A, T, G, or C, to DNAs of 4 or more 
different depots, the hybridization step would need to be repeated with 
each of the 4 different .epsilon..sub.X strands at the desired substrate 
locations prior to ligating and cleaving. After the first step in which an 
.epsilon..sub.X strand is hybridized to the substrate-bound DNA, and 
before each subsequent .epsilon..sub.X addition step, undesired 
.epsilon..sub.X strands are melted away from the DNA of the depots where 
the nucleotides are to be added by local heating, e.g., by using laser 
illumination patterned with a lithographic mask for 10 seconds to give a 
local temperature of approximately 70.degree. C., thereby producing the 
same duplex DNA structure comprising a duplex .alpha.-.beta. portion 
produced in Step 2, wherein .gamma. DNA strands in the selected areas are 
receptive to one of the .epsilon..sub.X oligomers. Desired .epsilon..sub.X 
strands are then hybridized to exposed .gamma. strands of substrate-bound 
DNA molecules at the heated locations by repeating Step 3. Local heating 
to selectively remove undesired .epsilon..sub.X strands without melting 
duplex .alpha.-.beta. portions (Step 4), and hybridization of desired 
.epsilon..sub.X strands at each location where nucleotides are to be added 
(Step 3), are repeated until all depots where nucleotides are to be added 
have been treated with a desired .epsilon..sub.X oligomer. 
Step (5): After all desired .epsilon. strands are hybridized to the growing 
DNA molecules, nicks between the 3' hydroxyl terminations of the .epsilon. 
strands and the 5' phosphate terminations of the .alpha. strands are 
ligated using T4 DNA ligase again. Ligation of the hybridized 
.epsilon..sub.X strands to the .alpha. strands by incubating with T4 DNA 
ligase and ATP gives: 
5' AGTGCAGTCTCGNTCTTAACATAGGAATTTGAGGCAGTACGCAAAAA 3'-B(.epsilon.+.alpha.) 
3' TCACGTCAGAGCNNNNNTTGTATCCTTAAACTCCGTCATGCGT 5' (.gamma.+.beta.), 
where the .gamma.+.beta. strand is SEQ ID NO: 11 as described above, and 
wherein and the .epsilon.+.alpha. strand is SEQ ID NO: 12 wherein N is A, 
C, G, or T. 
Step (6): The DNA-covered substrate is incubated at 37.degree. C. with Alw 
26 I restriction enzyme, and a small sticky-ended double-stranded oligomer 
is cut off and washed away producing: 
5' NTCTTAACATAGGAATTTGAGGCAGTACGCAAAAA 3'-B (.alpha.+N) 
3' NTTGTATCCTTAAACTCCGTCATGCGT 5' (.beta.+N), 
where .alpha.+N is SEQ ID NO: 13 and .beta.+N is SEQ ID NO:14, 
wherein N is A, C, G, or T, and 
5' AGTGCAGTCTCG 3' (SEQ ID NO: 15) 
3' TCACGTCAGAGCNNNN 5' (SEQ ID NO: 16), 
wherein the first N in the 3'.fwdarw.5' direction is a universal base and 
the subsequent N's designate A, C, G, or T. 
This leaves the new deoxyribonucleotides X of .epsilon..sub.X added to the 
.alpha. strands, and the .alpha.-.beta. strands in a state precisely like 
that encountered in step 2, except for being one base pair longer. 
Step (7): The synthetic cycle is now repeated by returning to Step 2 of the 
above-described example. By repeating Steps 2-6 one may now add as many 
bases as desired in what ever pattern is needed. 
Step (8). When the .alpha. strands have the desired sequence, the 
lengthened .beta. strands are melted off and washed away. The .beta.' 
strands are then allowed to hybridize with the .alpha. strands, leaving 
the newly synthesized oligonucleotides in single-strand form, attached at 
their 3' ends to the blunt-ended .alpha.-.beta. duplexes at the 5' ends of 
the .alpha. strands. If the number of added nucleotides happens to be 
four, this last step is not needed. 
The .epsilon. strands are more than 50% G-C's and would be expected to 
dissociate at a rate of less than 10.sup.-4 s.sup.-1 or less at 22.degree. 
C., compared to 10.sup.2 s.sup.-1 at 70.degree. C. (from extrapolation of 
the data in FIG. 6 of Morrison et al., Biochemistry, 1993, vol. 32, pages 
3095-3014; see also C. Cantor and P. Schimmel, Biophysical Chemistry, 
1980, Freeman Press, New York, page 1217). The melting point for similar 
14-mers is about 40.degree. C. (Wallace et al., Nucleic Acid Research, 
1979, vol. 6, pages 3543-3557). Thus, a 10 sec heat pulse raising the 
temperature of a spot to 70.degree. C. will result in a 99.9% chance that 
a new base is incorporated where it is wanted and a similar chance that it 
is not incorporated elsewhere if the temperature there is less than 
20.degree. C. The .beta.' strands dissociate at a rate of roughly 
10.sup.-5 s.sup.-1 at 70.degree. C., and thus the structure should be 
quite stable under the temperature cycling needed for Steps 2-7. 
It is estimated that when one depot is heated to the 70.degree. C. required 
in Step 4 the temperatures of any unilluminated neighboring depots will 
not rise above 20.degree. C. if the substrate is heat-sunk to near 
0.degree. C. Although the dissociation rates for oligomers are a steep 
function of temperature, the borders of the depots will contain sequences 
that do not correspond to the programmed growth. In operation, the area 
between the depots should not be subjected to heating; e.g., through use 
of a mask. 
At concentrations of 10.sup.-6 M (moles per liter), hybridization reactions 
rates are of the order of 1 s.sup.-1. The rate limiting steps in this 
scheme are the two ligation steps and the one restriction step. According 
to their catalog, one New England Biolabs (NEB) unit for the T4 DNA ligase 
gives 50% ligation of Hind III fragments in 30 m at a 5' DNA termini 
concentration of 10.sup.-7 M. Using a high concentration of enzyme will 
result in sufficiently complete ligation in a few minutes. The restriction 
enzyme will also act in a few minutes. The ligation and restriction enzyme 
cleavage steps need to occur only once in the four cycles. One may thus 
estimate that growth of a DNA array by the method described would take 
less than 30 minutes per four bases added, comparable to the 1 hour per 
four bases in the early light-directed synthesis work of Ref. 7. 
Example 2 
Releasing a set of oligonucleotides of known sequence from an 
oligomer-storing device 
This example, illustrated in FIGS. 5A and 5B, demonstrates an embodiment of 
the invention in which three selected DNA oligonucleotides, a, b, and c, 
are released from an oligomer storage device, and oligomers a and b are 
hybridized end-to-end to complementary oligomer c and are ligated together 
to produce a longer DNA molecule. This method is useful, for example, as a 
step in a protocol for solving a Hamiltonian path problem (Adleman et al., 
Supra, pages 1022-1023), or for making a synthetic gene. 
The device that stores and releases the oligomers comprises a 1 cm.times.1 
cm wafer of crystalline Al.sub.2 O.sub.3 substrate ((1) in FIG. 5A) that 
supports a square array of 165.times.165 depot pads. Wafers of crystalline 
Al.sub.2 O.sub.3, "synthetic sapphire", which are suitable for use with 
the present invention can be obtained from Saphikon, Milford, N.H., 03055. 
The top surface of each depot pad is 50 .mu.m.times.50 .mu.m, and the 
depot pads are spaced 10 .mu.m apart in both x and y directions in the 
array. Each depot pad comprises 3 layers, (1) a thermally insulating 1 
.mu.m thick layer of porous SiO.sub.2 which is attached to the Al.sub.2 
O.sub.3 substrate, (2) a light-absorbing 0.5 .mu.m thick layer of 
amorphous SiO.sub.2, and (3) a top, 0.5 .mu.m thick layer of SiO.sub.2, to 
which oligomers having selected nucleotide sequences are attached ((2) in 
FIG. 5A; see (10), (11), and (12) in FIG. 2). The attached oligomers are 
20-mer DNA oligonucleotides (20 nucleotides in length) that are covalently 
attached at their 3' ends to uncharged spacer groups, which spacer groups 
are covalently attached to the upper SiO.sub.2 surfaces of the depot pads. 
20-mer DNA oligonucleotides which are complementary to the attached 
oligomers are stored in the device by their being specifically hybridized 
to the attached oligomers by Watson-Crick base-pairing. The stored 
oligonucleotides have 5'-phosphate and 3'-OH termini, so that they can be 
ligated together. 
The depot array is immersed in about 100 .mu.l of solution containing 1 M 
NaCl, 5 mM EDTA, 0.1 M Tris-Cl, pH 8.0, 0.5% SDS. 
As shown in FIG. 5A, the depot sites storing oligonucleotides a, b, and c, 
are each irradiated through the Al.sub.2 O.sub.3 substrate with 
approximately 100 milliwatts of argon laser light (488 nm) ((9) in FIG. 
5A) to melt double-stranded oligonucleotide complexes at the heated depots 
and release the desired single-stranded DNA oligonucleotides molecules 
into the solution ((5) in FIG. 5). 
The 3' half of oligomer a and the 5' half of oligomer b are complementary, 
respectively, to the 3' and 5' halves of oligomer c. Thus as shown in FIG. 
5B, oligomer c hybridizes to the 3' end of oligomer a, and also to the 5' 
end of oligomer b, and it functions as a molecular splint by aligning the 
a and b oligomers end-to-end so that they can be covalently joined by 
ligase enzyme to produce a longer DNA molecule. 
While the invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications and this application is intended to cover any variations, 
uses, or adaptations of the invention following, in general, the 
principles of the invention and including such departures from the present 
disclosure as come within known or customary practice within the art to 
which the invention pertains and as may be applied to the essential 
features hereinbefore set forth and as follows in the scope of the 
appended claims. 
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# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 16 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - TCTTAACATA GGAATTTGAG GCAGTACGCA AAAA - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - TGCGTACTGC CTCAAATTCC TATGTTAAGA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - TGCGTACTGC CTCAAATTCC TATGTT - # - # 
26 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - NNNNNCGAGA CTGCACT - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - AGTGCAGTCT CGA - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - AGTGCAGTCT CGT - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - AGTGCAGTCT CGG - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - AGTGCAGTCT CGC - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 base p - #airs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - NNNGTCTCN - # - # 
- # 9 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - NNNNNGAGAC NNN - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 43 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - TGCGTACTGC CTCAAATTCC TATGTTNNNN NCGAGACTGC ACT - # 
- # 43 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - AGTGCAGTCT CGNTCTTAAC ATAGGAATTT GAGGCAGTAC GCAAAAA - # 
47 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - NTCTTAACAT AGGAATTTGA GGCAGTACGC AAAAA - # - 
# 35 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - TGCGTACTGC CTCAAATTCC TATGTTN - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - AGTGCAGTCT CG - # - # 
- # 12 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - NNNNCGAGAC TGCACT - # - # 
- # 16 
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