Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-30
2004-12-14
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S007100
Reexamination Certificate
active
06830902
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for increasing sensitivity and specificity of nucleic acid synthesis by reducing nonspecific nucleic acid synthesis occurring at ambient temperature. The invention also relates to novel nucleic acids which have high affinity to polymerases. The methods and compositions of the present invention can be used in DNA sequencing, amplification reactions, nucleic acid synthesis and cDNA synthesis.
The invention also relates to nucleic acids and compositions which are capable of inhibiting or preventing nucleic acid synthesis, sequencing, amplification and cDNA synthesis, for example, by binding one or more polypeptides with polymerase activity. In addition, the materials and methods of the present invention may be used as therapeutics to inhibit the replication of organisms that rely upon a reverse transcriptase activity for completion of their life cycle, such as retroviruses. The invention also relates to vectors and host cells comprising such nucleic acid molecules. The invention also concerns kits comprising the compositions or nucleic acids of the invention.
BACKGROUND OF THE INVENTION
DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon hybridization of a primer to the single-stranded DNA template, polymerases synthesize DNA in the 5′ to 3′ direction, successively adding nucleotides to the 3′-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded DNA template, can be synthesized.
Both mesophilic and thermophilic DNA polymerases are used to synthesize nucleic acids. Using thermostable rather than mesophilic polymerases is preferable since the higher annealing temperatures used with thermostable polymerases result in less non-specific DNA amplification from extension of mis-annealed primers. Even with thermostable polymerases, however, some primer sequences and certain experimental conditions can result in the synthesis of a significant amount of non-specific DNA products. These non-specific products can reduce the sensitivity of polymerase-based assays and can require extensive optimization for each primer set. In addition, this problem is intensified when polymerases having a high level of activity at ambient temperature are employed (for example, DNA polymerase from
Thermotoga neapolitana
).
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework 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 and RNA which the gene encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the “coding” strand) is produced by polymerase enzymes, resulting in a specific sequence of ribonucleic acid (RNA). This particular type of RNA, since it contains the genetic message from the DNA for production of a protein, is called messenger RNA (mRNA).
Within a given cell, tissue or organism, there exist many 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 full functional genetic content of a cell, tissue or organism.
A 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 chromatography matrices, such as cellulose or agarose, to which oligomers of thymidine (T) have been complexed. Since the 3′ termini on most eukaryotic mRNA molecules contain a string of Adenosine (A) bases, and since A binds to T, 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 (RT) or DNA polymerases having RT activity, 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 (i.e., a double-stranded cDNA) of the original mRNA (and thus of the original double-stranded DNA sequences, 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 vector, which is then introduced into a host bacterial, yeast, animal or plant cell, a process referred to as transformation or transfection. The host cells are then grown in culture media, resulting in a population of host cells containing (or in many cases, expressing) the gene of interest or portions of the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a vector (e.g., plasmid, viral vector, cosmid, etc.) to growth of host cell populations containing the isolated gene or gene portions, is termed “cDNA cloning.” If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a “cDNA library,” an appropriate term since the set of cDNAs represents a “population” of genes or portions of genes comprising the functional genetic information present in the source cell, tissue or organism.
Synthesis of a cDNA molecule initiates at or near the 3′ termini of the mRNA molecules and proceeds in the 5′-to-3′ direction successively adding nucleotides to the growing strand. Priming of the cDNA synthesis at the 3′-termini at the poly A tail using an oligo (dT) primer ensures that the 3′ message of the mRNAs will be represented in the cDNA molecules produced. The ability to increase sensitivity and specificity during cDNA synthesis provides more representative cDNA libraries and may increase the likelihood of the cDNA library having full-length cDNA molecules (e.g., full-length genes). Such advances would greatly improve the probability of finding full-length genes of interest.
In addition to their importance for research purposes, reverse transcriptase enzymes play a critical role in the life cycle of many important pathogenic viruses, in particular, the human immunodeficiency viruses (HIV). In order to complete its life cycle, HIV and other similar viruses must use a reserve transcriptase enzyme to convert the viral RNA genome into DNA for integration into the host's genomic material. Since this step is critical to the viral life cycle and host cells do not have any similar requirement for reverse transcriptase activity, the reverse transcriptase enzyme has been intensively studied as a chemotherapeutic target. In general, the bulk of therapeutic reagents directed at the reverse transcriptase enzyme have been nucleotide analogues, for example AZT. Other therapeutic modalities using oligonucleotide-based reagents, e.g., anti-sense oligonucleotides and ribozymes, have been used to inhibit viral replication, however, these reagents are not targeted specifically against reverse transcriptase activity, instead of being targeted against the nucleic acid of the viral genome. See, for example, Goodchild, et al., “Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides,”
Proc. Natl. Acad. Sci. USA
85:5507-5511 (1988), Matsukara, et al., “Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells,”
Proc. Natl. Acad Sci. USA
86:4244-4248 (1989), Rossi, et al., “Ribozymes as Anti-HIV-1 Therapeutic Agents: Pr
Astatke Mekbib
Chatterjee Deb K.
Gerard Gary F.
Horlick Kenneth R.
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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