Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-12
2004-09-07
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S025300
Reexamination Certificate
active
06787305
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the fields of molecular and cellular biology. The invention is related generally to compounds, compositions and methods useful in enhancing synthesis of nucleic acid molecules, especially from GC-rich nucleic acid templates. Specifically, the invention provides compositions comprising one or more compounds having a formula selected from the group consisting of formula I and formula II. Preferably used in accordance with the invention are 4-methylmorpholine N-oxide, betaine (carboxymethyltrimethyl ammonium), any amino acid (or derivative thereof, and/or an N-alkylimidazole such as 1-methylimidazole or 4-methylimidazole. In a preferred aspect, two or more, three or more, four or more, etc. of the compounds of the invention are combined to facilitate nucleic acid synthesis.
The invention also relates to compositions comprising one or more compounds of the invention and one or more additional components selected from the group consisting of (i) one or more nucleic acid molecules (including nucleic acid templates), (ii) one or more nucleotides, (iii) one or more polymerases or reverse transcriptases, and (iv) one or more buffering salts.
These compounds and compositions of the invention may be used in methods for enhanced, high-fidelity synthesis of nucleic acid molecules, including via amplification (particularly PCR), reverse transcription, and sequencing methods. The invention also relates to nucleic acid molecules produced by these methods, to fragments or derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules, fragments, or derivatives. The invention also relates to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising the compositions or compounds of the invention.
2. Related Art
Genomic DNA
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework (i.e., the genome) of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene ultimately encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the “sense” strand) is produced by polymerase enzymes, resulting in a specific sequence of messenger ribonucleic acid (mRNA). This mRNA is then translated by the protein synthesis machinery of the cell, resulting in the production of the particular protein encoded by the gene. There are additional sequences in the genome that do not encode a protein (i.e., “noncoding” regions) which may serve a structural, regulatory, or unknown function. Thus, the genome of an organism or cell is the complete collection of protein-encoding genes together with intervening noncoding DNA sequences. Importantly, each somatic cell of a multicellular organism contains the full complement of genomic DNA of the organism, except in cases of focal infections or cancers, where one or more xenogeneic DNA sequences may be inserted into the genomic DNA of specific cells and not into other, non-infected, cells in the organism. As noted below, however, the expression of the genes making up the genomic DNA may vary between individual cells.
cDNA and cDNA Libraries
Within a given cell, tissue or organism, there exist myriad mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cell—mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the fill functional genetic content of a cell, tissue or organism.
One common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs solid chromatography matrices, such as cellulose or hydroxyapatite, to which oligomers of deoxythytnidine (dT) have been complexed. Since the 3′ termini on all eukaryotic mRNA molecules contain a string of deoxyadenosine (dA) bases, and since dA binds to dT, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase, which results in the production of single-stranded cDNA molecules. The single-stranded cDNAs may then be converted into a complete double-stranded DNA copy of the original mRNA (and thus of the original double-stranded DNA sequence, encoding this mRNA, contained in the genome of the organism) by the action of a DNA polymerase. The protein-specific double-stranded cDNAs can then be inserted into a plasmid, which is then introduced into a host bacterial cell. The bacterial cells are then grown in culture media, resulting in a population of bacterial cells containing (or in many cases, expressing) the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a plasmid to growth of bacterial populations containing the isolated gene, is termed “cDNA cloning.” If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a “cDNA library,” representing the different functional (i.e., expressed) genes present in the source cell, tissue or organism. Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived.
DNA Amplification
In order to increase the copy number of, or “amplify,” specific sequences of DNA in a sample, investigators have relied on a number of amplification techniques. A commonly used amplification technique is the Polymerase Chain Reaction (“PCR”) method described by Mullis and colleagues (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159). This method uses “primer” sequences which are complementary to opposing regions on the DNA sequence to be amplified. These primers are added to the DNA target sample, along with a molar excess of nucleotide bases and a DNA polymerase (e.g., Taq polymerase), and the primers bind to their target via base-specific binding interactions (i.e., adenine binds to thymine, cytosine to guanine). By repeatedly passing the reaction mixture through cycles of increasing and decreasing temperatures (to allow dissociation of the two DNA strands on the target sequence, synthesis of complementary copies of each strand by the polymerase, and re-annealing of the new complementary strands), the copy number of a particular sequence of DNA may be rapidly increased.
Other techniques for amplification of target nucleic acid sequences have also been developed. For example, Walker et al. (U.S. Pat. No. 5,455,166; EP 0 684 315) described a method called Strand Displacement Amplification (SDA), which differs from PCR in that it operates at a single temperature and uses a polymerase/endonuclease combination of enzymes to generate single-stranded fragments of the target DNA sequence, which then serve as templates for the production of complementary DNA (cDNA) strands. An alternative amplification procedure, termed Nucleic Acid Sequence-Based Amplification (NASBA) was disclosed by Davey et al. (U.S. Pat. No. 5,409,818; EP 0 329 822). Similar to SDA, NASBA employs an isothermal reaction, but is based on the use of RNA primers for amplification rather than DNA primers as in PCR or SDA. Another known amplification procedure includes Promoter Ligation Activated Transcriptase (LAT) described by Berninger et al. (U.S. Pat. No. 5,194,370).
PCR-based DNA Fingerprinting
Despite the availability of a variety of amplification techniques, most DNA fingerprinting methods rely on PCR for amplification, taking ad
Gebeyehu Gulilat
Jessee Joel A.
Li Wu-Bo
Schuster David
Xia Jiulin
Riley Jezia
Sterne Kessler Goldstein & Fox P.L.L.C.
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