Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-10
2001-10-23
Zitomer, Stephanie W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S283100, C422S068100, C424S001730, C536S022100, C536S024300
Reexamination Certificate
active
06306587
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention pertains to methods, reagents, compositions, kits, and apparatus for use in ligating substantially contiguous ligands together on a target template. In particular, the present invention relates to methods, reagents, compositions, and kits for performing deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) hybridization assays.
The following definitions are provided to facilitate an understanding of the present invention.
The term “target” or “target molecule” refers to a molecule of interest, i.e. the molecule whose presence one wishes to know. The target is a member of a biological binding pair.
The term “biological binding pair” as used in the present application refers to any pair of molecules which exhibit mutual affinity or binding capacity. For the purposes of the present application, the term “ligand” will refer to one molecule of the biological binding pair, and the term “antiligand” or “receptor” will refer to the opposite molecule of the biological binding pair. For example, without limitation, embodiments of the present invention have application in nucleic acid hybridization assays where the biological binding pair includes two complementary strands of polynucleic acid. One of the strands is designated the ligand and the other strand is designated the antiligand or receptor. One of the strands may also be a target molecule. The designation of ligand or antiligand is a matter of arbitrary convenience. The biological binding pair may include antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates, to name a few. A biological binding pair is capable of forming a complex under bindings conditions.
The term “probe” refers to a ligand of known qualities capable of selectively binding to a target antiligand or receptor. As applied to nucleic acids, the term “probe” refers to a strand of nucleic acid having a base sequence complementary to a target strand. The probe and the target are capable of forming a probe target complex under binding conditions.
The term “label” refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes. The term “agent” is used in a broad sense, including any molecular moiety which participates in reactions which lead to a detectable response. The term “cofactor” is used broadly to include any molecular moiety which participates in reactions with the label.
The term “amplify” is used in the broad sense to mean creating an amplification product, which may include by way of example, additional target molecules, or target-like molecules, capable of functioning in a manner like the target molecule, or a molecule subject to detection steps in place of the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a polynucleotide, additional target, or target-like molecules, or molecules subject to detection can be made enzymatically with DNA or RNA polymerases.
The term “reactive functional group” refers to a functional group capable of forming a covalent bond upon activation between two ligands held in a reactive position. Activation may include chemical or physical changes to the environment.
The term “photoreactive functional group” refers to a reactive functional group capable of forming a covalent bond upon photoactivation with radiant energy between two ligands held in a reactive position.
An example of a photoreactive functional group includes, without limitation, olefins, conjugated olefins, ketones, &agr;, &bgr;-unsaturated ketones, azides, conjugated polyolefins characterized by conjugated double bonds and ketone functionality and aromatic compounds. Photoreactive functional groups can be further characterized as coumarins, psoralens, anthracenes, pyrenes, carotenes, tropones, chromones, quinones, maleic anhydride, alkyl maleimide and derivatives thereof. Further examples of photoreactive functional groups can be found in the reference J. G. Calvert, James N. Pitts, Jr.,
Photochemistry
, pages 536-48, John Wiley Sons, Inc. (1966), incorporated by reference herein.
The term “contiguous” means an adjacent area of a molecule. By way of example, in the case of biological binding pairs, where a first ligand binds to a receptor target molecule, the area surrounding and adjacent to the first ligand is open and capable of binding to a second ligand contiguous to the first. In the context of polynucleotides, where a first probe binds to an area of a polynucleotide target molecule, an adjacent mutually exclusive area along the length of the target molecule can bind to a second probe which will then be contiguous to the first. The target molecule acts as a template, directing the position of the first probe and the second probe. The term “substantially contiguous” is used in the functional sense to include spatial orientations which may not touch, may not abut, or may overlap yet function to bring a reactive covalent functional group into a reactive position.
The term “capture ligand” means a ligand capable of specifically binding with a capture antiligand associated with a support.
The term “retrievable support” is used in a broad sense to describe an entity which can be substantially dispersed within a medium and removed or separated from the medium by immobilization, filtering, partitioning, or the like.
The term “support”, when used alone, includes conventional supports such as filters and membranes as well as retrievable supports.
The term “reversible”, in regard to the binding of ligands and antiligands, means capable of binding or releasing upon imposing changes which do not permanently alter the gross chemical nature of the ligand and antiligand. For example, without limitation, reversible binding would include such binding and release controlled by changes in pH, temperature, and ionic strength which do not destroy the ligand or antiligand.
Genetic information is stored in living cells in thread-like molecules of DNA. In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding and &pgr;-stacking interactions. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
The genetic code of a living organism is carried upon the DNA strand in the sequence of base pairs. DNA consists of covalently linked chains of deoxyribonucleotides and RNA consists of covalently linked chains of ribonucleotides.
Each nucleic acid is linked by a phosphodiester bridge between the 5′-hydroxyl group of the sugar of one nucleotide and the 3′-hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a free 5′-hydroxyl group and another terminal end having a 3′-hydroxyl- group. The terminal ends of polynucleotides are often referred to as being 5′-termini or 3′-termini in reference to the respective free hydroxyl group. Naturally occurring polynucleotides may have a phosphate group at ,the 5′-terminus. Complementary strands of DNA and RNA form antiparallel complexes in which the 3′-terminal end of one strand is oriented and bound to the 5′-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at their complementary regions. Presently, nucleic acid hybridization assays are primarily used to detect and identify unique DNA or RNA base sequences or sp
Cruickshank Kenneth A.
Morrison Larry E.
Royer Garfield P.
Murray William E.
Tung Joyce
Vysis, Inc.
Zitomer Stephanie W.
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