Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1994-04-14
2002-12-24
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S091210, C435S005000, C435S006120, C435S455000, C435S471000, C435S320100, C435S252300, C536S023100, C536S024100, C536S024330, C536S023500
Reexamination Certificate
active
06498025
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and compositions for DNA synthesis, and, more particularly, for the synthesis of complementary DNA in vivo.
BACKGROUND OF THE INVENTION
The present invention is a tool for molecular biology. An introduction to the nomenclature of molecular biology, the structure of DNA, RNA and proteins and the interrelationships between these molecules, is provided in Chapter 4
, Synthesis of Proteins and Nucleic Acids
of Darnell et al.,
Molecular Cell Biology
, Scientific American Books (1989). A more detailed treatment of these issues is set forth in the full text of Darnell et al., (1989) and in Lewin,
Genes IV
, Oxford University Press (1990).
Hereditary information is encoded in the genes of an organism. Genes are made of polymers of nucleic acids, usually deoxyribonucleic acid (DNA). DNA is composed of a series of four nucleotide bases; the hereditary information carried by a gene is encoded by the specific sequence of nucleotide bases in the DNA molecule. The genetic information within structural genes encodes proteins; the sequence and structure (and therefore function) of a particular protein is determined by the order of the nucleotide bases within the gene that encodes that protein. Proteins determine an organism's identity; from cellular structures to the organism's response to its environment. Thus, the genes that encode these proteins determine an organism's identity.
The information encoded within a structural gene is “expressed” by a cell through the processes of transcription and translation. Transcription results in the production of an intermediate carrier of the genetic code, termed messenger RNA (mRNA). Messenger RNA is effectively a copy of the gene; it is a polymer of ribonucleic acid (hence “RNA”) rather than of deoxyribonucleic acid.
In eukaryotic organisms (which are generally more complex organisms than bacteria), genes are made up of coding regions (termed “exons”) and non-coding regions (termed “introns”). Exons directly encode the protein sequence of the gene. Introns may be very large and there may be a large number of intron sequences within a particular gene. The role of the non-coding intron sequences is unclear. However, there is evidence that these intervening sequences serve two critical purposes: first they divide the exon coding regions into smaller protein coding units and so minimize the chances of errors during transcription and translation; second, they relegate discrete portions or cassettes of protein sequence to exon units which can be more easily shuffled during the course of evolution and therefore facilitate the development of new proteins which may ultimately enhance the survival of the species.
The transcription process involves the formation of an mRNA copy of the entire gene. That is, the mRNA produced by the transcription process contains a copy of both the non-coding intron sequences and the protein-encoding exon sequences. Thus the mRNA first produced by transcription is the same length as the gene from which it was copied. Subsequently, this immature mRNA undergoes a processing stage during which the non-coding intron sequences are spliced out. The resulting processed mRNA molecules thus contain only the information required to encode the protein (i.e. they contain copies of only the joined exon sequences). These processed mRNA molecules are thus considerably shorter in length than the “genomic sequence” (the gene exons and introns as they exist in the chromosome) from which the mRNA was initially copied. The processed mRNA is also modified at this stage to include a polyriboadenylic acid, poly(A), tail at one end of the molecule (the 3′ end) and a “cap” structure at the other end of the molecule (the 5′ end) (standard nomenclature assigns one end of DNA and RNA molecules as the 5′ end and the other as the 3′ end, according to the terminal chemical groupings of the molecule). An mRNA molecule that has been processed to remove introns and has a 5′ cap and a 3′ poly(A) tail is termed a “mature” mRNA molecule. A greatly simplified diagram of the transcription process, illustrating removal of the non-coding intron sequences is shown in FIG.
1
.
The step of converting the information carried by the mature messenger RNA into a protein is termed translation. Translation is the final step of the means by which the information encoded by the nucleotide sequence within a structural gene is converted into a specific protein composed of a sequence of amino acids.
The cloning of genes became possible in the 1970's. In early experiments, small genes were cloned from bacteria. Since that time advances in molecular biology and genetic engineering have developed at an extraordinary rate, such that the sequence of the entire human genome is now being determined. Despite rapid advances in the technology of this field, a number of limitations are still apparent. One of these is the difficulty of cloning very large structural genes.
The size of a gene is measured in the number of nucleotide bases that it contains, usually expressed in terms of thousands of bases (kilobases or Kb). Although there are several examples of larger genes, the total coding sequence of most structural genes (the exons) typically totals 1-10 Kb. However, the presence of multiple large intron sequences between the exon segments means that at the genomic level these genes are spread out over a much larger area, frequently spanning tens or even hundreds of kilobases. Present gene cloning vectors such as YACs (Yeast Artificial Chromosomes) allow the cloning of very large (100-300 Kb) genomic segments; however, these genomic inserts include the noncoding intron sequences, which precludes the expression of protein in an artificial system. A partial genetic sequence, or sequence containing introns, results in the expression of a nonfunctional, truncated protein, or, when the sequence for the 5′ translation start site is missing, results in expression of a unrelated garbled protein sequence. Even if a partial gene may be identified through a screening process, it is then necessary to recover the remaining portions of the gene. This can be an extremely complicated process. If the gene contains many intron sequences, and is thus large, years of effort can be expended in attempting to recover the remaining pieces of the gene. Additional effort may then be required to determine the relative order of the gene fragments and to distinguish exon from intron sequences. The ability to clone a gene as a contiguous protein coding cassette is particularly important where identification of the gene is achieved by means of a detection technique which relies on production of the protein in a recombinant bacterial or viral system and “screening” for the function or structure of the desired protein—a common technique of detecting cloned genes.
To clone structural genes, molecular biologists have taken advantage of the cellular mRNA processing function described above whereby intron sequences are spliced out of the immature mRNA to produce a mature mRNA that is considerably smaller that the original gene. By converting the mature mRNA molecule back into a DNA molecule (hence the term, “reverse transcription”), one can obtain the original coding sequence (the exons) without the extraneous intron sequences. Such a DNA molecule is termed a complementary DNA because it is complementary to the mRNA molecule from which it was derived. Complementary DNA (cDNA) synthesis is the preferred technique for gene cloning because it results in the recovery of the desired gene in a relatively small, contiguous protein coding cassette amenable to recombinant protein production.
An additional and important use of cDNA technology is to identify those genes that are being expressed by a cell at a particular time. Gene expression requires substantial energy expenditure on the part of the cell, and mRNA molecules are designed to be short-lived “protein requests”; therefore, with a few exceptions (notably in th
Myers Carla J.
The Law Offices of James C. Weseman
Weseman, Esq. James C.
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