Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-08-05
2001-12-04
Campbell, Eggerton A. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120
Reexamination Certificate
active
06326489
ABSTRACT:
BACKGROUND OF THE INVENTION
Compact arrays or libraries of surface-bound, double-stranded oligonucleotides are of use in rapid, high-throughput screening of compounds to identify those that bind, or otherwise interact with, short, double-stranded DNA sequence motifs. Of particular interest are proteins, particularly trans-regulatory factors, that control gene transcription. Ideally, such an oligonucleotide array is bound to the surface of a solid support matrix that is of a size that enables laboratory manipulations, e.g. an incubation of a candidate protein with the nucleic acid targets sequences thereon, and that is itself inert to chemical interactions with experimental proteins, buffers and/or other components. In addition, it is desirable that the absolute number of unique target sequences in the array be maximized, since methods of high-throughput screening are used in the attempt to minimize repetition of steps that are labor-intensive or otherwise costly.
A high-density, double-stranded DNA array complexed to a solid matrix is described by Lockhart (U.S. Pat. No.: 5,556,752); however, the DNA molecules therein disclosed are produced as unimolecular products of chemical synthesis. Each member of the array contains regions of self-complementarity separated by a spacer (i.e. a single-strand loop), such that these regions hybridize to each other in order to produce a double-helical region. A difficulty of such a production method arises when the accuracy of chemical synthesis is considered in light to that of that demonstrated by proteinaceous DNA polymerase molecules. It is estimated that enzymatic synthesis of second-strand DNA from a first-strand template operates at 100-fold higher fidelity than do chemical synthetic procedures. Further, it is required that those regions of complementary nucleic acid sequences that must hybridize in order to form the double-helical structure are physically attached to each other by a linker subunit.
SUMMARY OF THE INVENTION
The present invention encompasses an array of surface-bound, bimolecular, double-stranded, nucleic acid molecules, the array comprising a solid support, and a plurality of different double-stranded nucleic acid molecule members, a member comprising a first nucleic acid strand linked to the solid support and a second nucleic acid strand which is substantially complementary to the first strand and complexed to the first strand by Watson-Crick base pairing, wherein at least a portion of the members have a second nucleic acid strand which is substantially complementary to and base paired with the first strand along the entire length of the first strand.
The term “synthetic”, as used herein, is defined as that which is produced by in vitro chemical or enzymatic synthesis. The synthetic arrays of the present invention may be contrasted with natural nucleic acid molecules such as viral or plasmid vectors, for instance, which may be propagated in bacterial, yeast, or other living hosts.
As used herein, the term “nucleic acid” is defined to encompass DNA and RNA or both synthetic and natural origin. The nucleic acid may exist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer, wherein the term “copolymer” refers to a single nucleic acid strand that comprises both ribonucleotides and deoxyribonucleotides.
The phrase “different nucleic acid molecule members” means that the double-stranded nucleic acid molecules attached to the surface include double-stranded nucleic acid molecules of of different nucleotide sequence.
When used herein in this context, the term “double-stranded” refers to a pair of nucleic acid molecules, as defined above, that exist in a hydrogen-bonded, helical array typically associated with DNA, and that under these umbrella terms are included those paired oligonucleotides that are essentially double-stranded, meaning those that contain short regions of mismatch, such as a mono-, di- or tri-nucleotide, resulting from design or error either in chemical synthesis of the oligonucleotide priming site on the first nucleic acid strand or in enzymatic synthesis of the second nucleic acid strand.
As used herein, the terms “complementary” and “substantially complementary” refer to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.
Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
As used herein, the term “array” is defined to mean a heterogeneous pool of nucleic acid molecules that is affixed to a substrate or solid support in a manner that permits identification of individual members during the course of experimental manipulation.
According to the invention, the array may have virtually any number of different members. In preferred embodiments, the array comprises from 2 up to 100 members, more preferably from 100 up to 10,000 members and highly preferably from 10,000 up to 1,000,000 members, preferably on a solid support. In preferred embodiments, the array will have a density of more than 100 members at known locations per cm
2
, preferably more than 1,000 per cm
2
, more preferably more than 10,000 per cm
2
.
According to the methods disclosed herein, a “substrate” or “solid support” is defined as any material having a rigid or semi-rigid surface.
It is contemplated that attached to the solid support is a spacer. The spacer molecule is preferably of sufficient length to permit the double-stranded oligonucleotide in the completed member of the array to interact freely with molecules exposed to the array. The spacer molecule, which may comprise as little as a covalent bond length, is typically 6-50 atoms long to provide sufficient exposure for the attached double-stranded DNA molecule. The spacer is comprised of a surface attaching portion and a longer chain portion.
Preferably, the 3′ end of the first strand is linked to the solid support.
It is preferred that the 5′ end of the first strand and 3′ end of the second strand are not linked via a covalent bond, and thus do not form a continuous single strand. As used herein in this context, “covalent bond” is defined as meaning a bond that forms, directly or via a spacer comprising nucleic acid or another material, a continuous strand that comprises the 5′ end of the first strand and the 3′ end of the second strand, and thus includes a 3′/5′ phosphate bond as occurs naturally in a single-stranded nucleic acid. This definition does not encompass intermolecular crosslinking of the first and second strands.
It is additionally preferred that the 5′ end of the second strand is not linked to the support.
It is preferred that the solid support is a silica support.
It is also preferred that the first strand is produced by chemical synthesis and that the second strand is produced by enzymatic synthesis.
Preferably, the first strand is used as the template on which the second strand is enzymatically produced.
It is additionally preferred that in each member of the array, the first strand contains at its 3′ end a binding site for an oligonucleotide primer which is used to prime enzymatic synthesis of the second strand, and at its 5′ end a variable sequence.
An “oligonucleotide primer”, as referred to herein, is defined as a single-stranded DNA or RNA molecule that is hybridized to a nucleic acid template to prime enzymatic synthesis of a second nucleic acid strand.
It is preferred that enzymatic synthesis of the second strand is performed using an enzyme. Preferably, the oligonucleo
Bulyk Martha L.
Church George M.
Banner & Witcoff , Ltd.
Campbell Eggerton A.
Howard Hughes Medical Institute
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