Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-16
2003-04-08
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091100, C435S091410, C435S091500, C435S091510, C435S091520, C536S023100, C536S024330
Reexamination Certificate
active
06544736
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for synthesizing cDNA from a mRNA sample and to tobacco acid pyrophosphatase for use in the method; more specifically, the invention relates to a rapid synthesis method of cDNA including the 5′-terminal region of mRNA in a sample for the analysis of a nucleotide sequence derived from the 5′-terminus of the mRNA.
2. Background of the Invention
Numerous types of proteins composing cell are present, such as proteins involved in cell morphology, proteins involved in development or proteins involved in metabolism. The patterns of the presence undoubtedly determine the properties of cell. Essential information relating to the mode of these presence and functions is imprinted in the gene in cell and is realized by mRNA as a copy of the gene. mRNA functions as template for protein translation and also as a carrier of the information flow from DNA to protein. Ultimately, mRNA reflects the “phenotypes” in all biological organisms. These proteins are industrially valuable and possibly applicable as pharmaceutical drugs, diagnostic agents, bio-sensors and bio-reactors, provided that these proteins are biologically active substances. Hence, it is very important to recover full-length mRNA and procure gene information from the mRNA. Recent progress in the gene recombinant technology and more recent promotion in the genome analysis project are now permitting cDNA cloning and analysis technology readily usable.
Although rapid analysis of complete 5″-terminal sequence of mRNA has increasingly been demanded in recent years, no technology has been established yet to enable such rapid analysis in a simple and rapid fashion. Because the 5′-terminal sequence of full-length mRNA contains a transcription start for gene expression analysis on genome, rapid analysis of the 5′-terminal sequence as well as enormous quantities of sequenced genome open up a way for transcription gene mapping. Furthermore, accurate information of the 5′-terminal sequence of mRNA can identify the sequences of gene expression regulatory promoters present upstream. These promoters are cis-factors regulating when, where and how much a gene should be expressed. The detection of the 5′-terminal sequence of mRNA verifies that an upstream promoter sequence is functional, which suggests a new possibility for the etiological analysis or diagnosis or therapeutic treatment of diseases.
Practically, the information as to when, where and how much a gene is expressed is very valuable information for the etiological analysis or diagnosis or therapeutic treatment of diseases. The Human Genome Project currently promoted internationally mentions as one of the goals to collect such information. The ultimate purpose of the Project lies in the nucleotide sequencing of biological genome. The nucleotide sequences of several bacterial genome species and the nucleotide sequences in the whole genome of budding yeast have already been sequenced and reported. Most of many genes identified on the isolated genome species are functionally not yet identified, which is a big issue in future. In that sense, the significance of the analysis of cDNA reflecting the gene expression dynamics in cell is increasingly drawing attention.
Herein, by the term cDNA referred to as complementary DNA is meant DNA synthetically prepared by reverse transcriptase using mRNA as template. In other words, the information of mRNA encoding the information of the amino acid sequence of protein is synthetically constructed as cDNA. The analysis of the cDNA can readily determine the primary structure of the protein and can readily promote the development of a large-scale expression system. Thus, such cDNA preparation is now very important, industrially.
Ideally, the ultimate goal of the cDNA cloning technology lies in the replacement of all expressed mRNAs with complete cDNAs. Thus, the information is greatly valuable. In other words, the information recovered from such full-length cDNA serves as a starting point for the analysis of the information on genome, because the information includes the information of transcription start and the entire information of expressed protein. The primary protein sequence recovered from a complete coding sequence distinctively shortens the time required for the functional analysis.
However, the technology for the recovery of cDNA including full-length mRNA has been a not-yet matured technology “still under way of development” among the DNA technologies in rapid progress. For example, the Gubler-Hoffman method (Gene, Vol. 25, pp. 236-269, 1983) is known as one of synthesis method of cDNAs commonly applied conventionally. Nevertheless, many of cDNAs synthesized by the method are incomplete with terminal deficiency. Alternatively, the Okayama-Berg method (Mol. Cell. Biol., Vol. 2, pp. 161-170, 1982) is a synthesis method characteristic in that full-length cDNA is readily prepared. Even by the method, however, reverse transcription sometimes stops in the course of cDNA synthesis, so no guarantee is given to the resulting cDNA that it is of full length.
The RACE method (Rapid amplification of cDNA ends: Proc. Natl. Aca. Sci. USA, Vol. 85, pp. 8998-9002, 1988) has been suggested as a method to supplement a portion lacking in cDNA, based on the partial cDNA sequences recovered by the existing methods, so as to acquire the complete information of mRNA. The method comprises reverse transcription based on a target cDNA sequence to add a homopolymer to both the ends of cDNA by terminal transferase or to ligate an adapter comprising a synthesized DNA to both the ends of cDNA by T4 DNA ligase, and polymerase chain reaction (PCR) based on these added sequences and a primer specific to the target cDNA, thereby analyzing only the terminal regions of mRNA sequence.
The analysis of the target 5′-terminus of mRNA in particular by the method (referred to as 5′-RACE) can be done in a very simple fashion, because PCR is utilized by the method. Accordingly, the method is frequently used. Principally, however, the method apparently cannot analyze the 5′-terminal sequence of mRNA used for the preparation of the cDNA, although the method can analyze the 5′-terminus of cDNA. Hence, the recovery of complete 5′-terminus of mRNA is very difficult, compared with the recovery of complete 3′-terminus by 3′-RACE, in which poly-A sequence is responsible for the protection role against terminal deficiency. As described above, even currently, the method is acclaimed as a “not-yet established technology”.
It is known that the 5′-terminus of complete mRNA has a characteristic structure called cap structure (Nature, Vol. 253, pp. 374-375, 1975). An attempt has been suggested to analyze cDNA, targeting the vicinity of the cap structure (Japanese Patent Laid-open No. 6-153953 (1994); Gene, Vol. 138, pp. 171-174, 1994).
According to these methods, tobacco acid pyrophosphatase (referred to as “TAP” hereinafter) specifically cleaving the cap structure is used. These methods comprise treating mRNA with alkali phosphatase to remove the phosphate group from the 5′-terminus of mRNA without any cap, subsequently treating the resulting mRNA with TAP to cleave the cap, adding an oligoribonucleotide and continuously effecting reverse transcription, to synthesize cDNA. Although these methods are complicated because enzymatic reactions continue over plural steps, these methods are a few effective methods principally capable of specifically analyzing full-length mRNA. Nevertheless, these methods include problems to be improved in the steps. Currently, therefore, these methods are not commonly widespread, although these methods are greatly needed due to the significance of the 5′-terminal sequencing as described above.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a synthesis method of cDNA from mRNA, so as to recover the complete 5′-terminal sequence of cDNA at a large-sca
Funaki Hiroko
Furuichi Yasuhiro
Ohara Eiji
Shibata Yuko
Shimamoto Akira
Frishauf Holtz Goodman & Chick P.C.
Jones W. Gary
Nippon Gene Co., Ltd.
Taylor Janell E.
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