Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1994-12-21
2001-03-27
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06207368
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the analysis of deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), the determination of the presence of specific DNA and/or RNA nucleotide sequences, and the exponential amplification of such sequences.
BACKGROUND OF THE INVENTION
The references to be discussed throughout this document are set forth solely for the information described therein prior to the filing date of this document, and nothing herein is to be construed as an admission, either express or implied, that the references are “prior art” or that the inventor is not entitled to antedate such descriptions by virtue of prior invention or priority based on earlier filed applications.
I. Introduction
The amplification of desired portions or entire sequences of DNA and RNA finds utility in a variety of fields, from criminal investigations (where DNA obtained from crime scene samples are compared with the DNA from an accused individual), to archeology (where the DNA of ancient plants, animals, sub-human species and humans are analyzed), to paternity analysis (where the DNA from the offspring and a possible parent are comparatively analyzed), to genetic analysis (where the DNA of individuals are analyzed for an indication of the possibility of genetic variation which is indicative of a particular disease state). Amplification of the nucleic acid sequence is most typically necessary because whatever DNA may be present from the source is extremely limited such that in order to properly analyze such DNA, many more copies of the original RNA are required.
The ability to amplify nucleic acid sequences is relatively recent (1985), but the impact of this ability has been phenomenal—without such amplification, most of the foregoing exemplary fields would not be possible. Thus, as the areas in which DNA amplification has expanded, the requirements placed upon various amplification techniques have changed. Accordingly, a very real and ongoing need exists for highly specific amplification techniques.
II. The Genetic Code
(a) Background Information
Deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), are long, thread-like macromolecules, DNA comprising a chain of deoxyribonucleotides, and RNA comprising a chain of ribonucleotides. A “nucleotide” consists of a nucleoside and one or more phosphate groups; a “nucleoside” consists of a nitrogenous base linked to a pentose sugar; a “pentose sugar” comprises five carbon atoms. In a molecule of DNA, the pentose sugar is “deoxyribose” and the nitrogenous base can be adenine (“A”), guanine (“G”), thymine (“T”) or cytosine (“C”). In a molecule of RNA, the pentose sugar is “ribose”, and the nitrogenous bases are the same as DNA, except uracil (“U”) replaces thymine. The specific sequence of the nitrogenous bases encodes genetic information, or, the “blueprint” for life.
Double stranded DNA consists of two “complementary” strands of nucleotide chains which are held together by (relatively) weak hydrogen bonds—these bonds can be “broken” by, e.g., heating the DNA, changing the salt concentration of a fluid surrounding the DNA, or chemical manipulation; this process is referred to as “denaturation”. By lowering the temperature, adjusting anew the salt concentration or removing
eutralizing the chemical, the two strands of DNA have a tendency to re-form in their approximate/identical original state. The bases of each DNA molecule selectively bind to each other: A always bonds with T, and C always bonds with G. Thus, the sequence “ATCG” of a first strand lies immediately opposite a complementary sequence “TAGC”. This is referred to as “complementary base pairing” and the process of complementary base paring is referred to as “hybridization”.
Three types of RNA (messenger RNA, mRNA; transfer RNA, tRNA; ribosomal RNA, rRNA) are associated with translation of the genetic information encoded in the DNA into designated amino acids, which are the building blocks for polypeptides and proteins; each of twenty naturally occurring amino acids is encoded by various groupings of three nucleotides, this grouping being referred to as a codon. Thus, the primary sequence of proteins are comprised of amino acids assembled in ribosomes based on codons defined by mRNA. Proteins are necessary to the development, maintenance and existence of living organisms; the presence, or absence, of certain proteins in different cells/tissues can be indicative of the presence, or absence of, e.g., certain biological functions of the aforementioned cells/tissues.
Genetic information is generally transferred as follows: DNA→RNA→amino acid/protein. Not every region of a DNA molecule is translated by RNA into protein; those regions that are translated are referred to as “genes.” Expression of genes, therefore, serves to control the transition of hereditary characteristics by specifying the eventual proteins produced from a gene, or genes.
(b) Mutations in the Genetic Code
DNA macromolecules are chemically quite similar to each other. A and G are quite similar in chemical composition, and C, T and U are equally similar. Thus, in a specified sequence, substitutions, e.g., transitions, of an A for a G or a C for a T may occur likewise, “transversions” of an A or G for a C or T (or vice versa) may occur. When such a substitution occurs within a codon such that the amino acid encoded thereby remains the same, then the substitution can be referred to as a “silent” substitution, i.e., the nucleotides are different but the encoded amino acid is the same. However, other substitutions can alter the amino acid encoded by the codon; when the nucleotide alteration results in a chemically similar amino acid, this is referred to as a “conservative” alteration, while a chemically different amino acid resulting from the alteration is referred to as a “non-conservative” alteration. Non-conservative alterations of amino acids can result in a molecule quite unlike the original protein molecule.
A protein that has had its amino acids altered can be referred to as a “mutant”, “mutation” or “variant.” Mutations occur naturally and can have positive, negative or neutral consequences on the organism experiencing such a mutation. Similarly, genes that have had sections altered (e.g., by insertion or deletion of DNA sequence(s) are mutations; thus, by definition, the proteins expressed by such a mutated gene can have positive, negative or neutral consequences on the organism.
By way of example, the gene responsible for the disease cystic fibrosis (a genetically inherited disorder affecting children and young adults and which is clinically manifested by the obstruction of the airways by thick, sticky mucus and subsequent infection) comprises 250,000 nucleotides, which encode a protein of 1480 amino acids (the protein is referred to as “cystic fibrosis transmembrane conductance regulator”, or “CFTR”). When this gene is compared with individuals who do not have the CF gene (i.e., individuals who have a “normal” gene), a frequent difference evidenced is that a single codon is deleted from the “normal” gene, which results in the loss of a single amino acid from the “normal” protein. However, this CFTR gene mutation accounts for only about 70% of those individuals who have CF; there are at least about 170 different CFTR gene mutations which account for the remaining 30%.
III. The Structural Formation of DNA/RNA Macromolecular Strands
While the sequence of the nitrogenous bases of the DNA and RNA macromolecule encode genetic information, the sugar and phosphate groups perform a structured role, forming the backbone of the molecule (typically, the phosphate group is attached to the fifth carbon , “C-5” or “5”, hydroxyl group (“OH”) of the pentose sugar). Specifically, a 3′-hydroxyl group of a first nucleotide is linked to a 5′-hydroxyl group of a second, adjacent nucleotide. The linkage between the two pentose sugars is via a phosphodiester bond. Based upon this linkage protocol, one end (“terminus”) of the nucleotide chain has a 5′-terminus and the other end has a 3′
Beckman Coulter Inc.
Grant Arnold
Houtteman Scott W.
May William H.
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