Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-18
2003-06-03
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C536S022100, C536S023100
Reexamination Certificate
active
06573048
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention relates to nucleic acid probes that are susceptible to chemical or enzymatic degradation and to assays and methods using such probes in the detection of target nucleic acid sequences in a sample.
b) Description of Related Art
The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the molecular repositories for genetic information. Ultimately, every protein is the result of the information contained in the cell's nucleic acids. A gene is a segment of DNA that contains the information for a functional biological product such as a protein or RNA. The function of DNA is the storage of biological information, and since cells have typically many thousands of genes, DNA molecules tend to be very large. The total length of all DNA in a single human cell is about two meters, consisting of billions of nucleotides.
In eukaryotic organisms, DNA is largely found in the nucleus of the cell. But protein synthesis occurs on ribosomes in the cytoplasm, hence a molecule other than DNA must carry the genetic message for protein synthesis from the nucleus to the cytoplasm. RNA is found in both the nucleus and cytoplasm, and it carries genetic information from DNA to the ribosome. Several classes of RNAs exist, each having distinct function. Ribosomal RNAs (rRNA) are structural components of ribosomes that carry out the synthesis of proteins. Messenger RNAs (mRNA) are nucleic acids that carry the information from the genes to the ribosome, where the corresponding proteins are made. Transfer RNAs (tRNA) are adapter molecules that translate the information in mRNA into a specific sequence of amino acids. There are also a wide variety of special-function RNAs that carry out additional functions in the cell (Lehninger et al.,
Principles of Biochemistry
, Second Edition, 1993, Worth Publishers, Inc.).
Double-helical DNA consists of two polynucleic acid strands, twisted around each other.
Each nucleotide unit of the polynucleotide strand consists of a nitrogenous base (A, T, C, or G), a sugar deoxyribose, and a phosphate group. The orientation of the two polynucleotide strands is antiparallel in that their 5′ to 3′ directions are opposite. The strands are held together by ydrogen bonds and hydrophobic interactions. The base pairs in DNA consist of purines such as adenine (A) and guanine (G), and pyrimidines such as thymine (T) and cytosine (C). A is paired with T by forming two hydrogen bonds while G is paired with C by forming three hydrogen bonds. This base pair complementarity is an essential feature of the DNA molecule and is due to the size, shape, and chemical composition of the bases. As a result of the geometry of the double helix, a purine must always pair with a pyrimidine. Furthermore, G will always pair with a C, and A will always pair with T. This simple and elegant structure provides for the extraordinary stability of the double helix.
Under the conditions of temperature and ion concentration found in cells, DNA is maintained as a two-stranded structure by the hydrogen bonds of the A-T and G-C base pairs. The duplexes can be melted (denatured into single strands) by heating them (usually in a dilute salt solution of, for example, 0.01 M NaCl) or by raising the pH above 11. If the temperature is lowered and the ion concentration in the solution is raised, or if the pH is lowered, the single strands will anneal, or reassociate, to reconstitute duplexes (if their concentration in solution is great enough). This property is the basis of a technique referred to as nucleic acid hybridization. In a mixture of nucleic acids, only complementary strands will reassociate; the extent of their reassociation is virtually unaffected by the presence of noncomplementary strands. The molecular hybridization can take place between complementary strands of either DNA or RNA or between an RNA strand and a DNA strand.
The use of various hybridization techniques employing oligonucleotide probes to detect genes (and RNA) of interest is well known in the art of molecular biology. Generally, probes are designed so that they hybridize to fragments containing a complementary nucleic acid sequence. The existence and amounts of hybrid formed are detected by measuring radiation (for radioactive probes), enzyme-produced products (for enzyme-labeled probes), fluorescence (for fluorescent-labled probes), and the like, depending on the nature of the signal being used. Various experimental conditions must be calculated to estimate nucleic acid duplex stability of probe-target complexes and to reduce nonspecific (background) binding of probes to non-target DNA or RNA. Due to the many variables that need to be considered when performing hybridization assays, including melting temperature or other denaturation conditions, annealing temperature, salt concentration, pH, and others, the likelihood of nonspecific binding of nucleic acid probes to nontarget nucleic acid sequences is still a major shortcoming when performing various hybridization techniques.
One use of molecular hybridization that has achieved prominence is in situ hybridization. abeled DNA or RNA that is complementary to specific sequences of DNA or RNA in a sample s prepared. Such complementary DNA or RNA is referred to as an oligonucleotide probe. In this assay, oligonucleotide probes are designed to anneal to specific target RNA such as mRNA, or particular native or integrated gene sequences in the DNA. Cells or tissue slices can be briefly exposed to heat or acid, which fixes the cell contents, including the nucleic acid, in place on a glass slide, filter, or other material. The fixed cell or tissue is then exposed to labeled complementary DNA or RNA probes for hybridization. Labeling agents may be radioisotopes such as
32
P, fluorescent dyes, biotinylated nucleotide analogues, antigens or any other commonly performed labeling technique. After a period of incubation, unhybridized labeled DNA or RNA can be removed while the hybridized complexes are detected to reveal the presence and/or location of specific RNA or DNA within individual cells or tissue slices. Although this technique is popular, continued efforts are necessary to improve the sensitivity of the assay and to decrease non-specific binding (background) of labeled probes (Damell et al.,
Molecular Cell Biology
, Second Edition, 1990, Scientific American Books, Inc.).
Another hybridization technique that is commonly used in the art is the hybridization of labeled probes to immobilized nucleic acids. There are many methods available to hybridize labeled probes to nucleic acids that have been immobilized on solid supports such as nitrocellulose filters or nylon membranes. These methods differ in various respects, such as solvent and temperature used; volume of solvent and length of hybridization; degree and method of agitation; use of agents such as Denhardt's reagent or BLOTTO to block the nonspecific attachment of the probe to the surface of the solid matrix; concentration of the labeled probe and its specific activity; use of compounds, such as dextran sulfate or polyethylene glycol, that increases the rate of reassociation of nucleic acids; and stringency of washing following the hybridization.
In traditional assay methods, several different types of agents can be used to block the nonspecific attachment of the probe to the surface of the solid support. Such agents include Denhardt's reagent, heparin, nonfat dried milk, and the like. Frequently, these agents are used in combination with denatured, fragmented salmon sperm or yeast DNA and detergents such as SDS. Blocking agents are usually also included in both the prehybridization and hybridization solution when nitrocellulose filters are used. When nylon membranes are used to immobilize the nucleic acids, the blocking agents are often omitted from the hybridization solution, since high concentrations of protein are believed to interfere with the annealing of the probe to its target. In order to minimize background problem
Albagli David
Cheng Peter C.
VanAtta Reuel
Wood Michael L.
Dorsey & Whitney LLP
Naxcor
Siew Jeffrey
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