Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1994-07-25
1997-01-28
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 915, 435 9151, 435 9152, 536 231, 536 2433, C12N 1511, C12N 1564, C12P 1934
Patent
active
055977138
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a process for synthesizing a full-length cDNA from mRNA. This invention enables screening of proteins useful in industry and mass production of the proteins.
BACKGROUND ART
Bio-active proteins produced by cells have been widely utilized in industry as materials for medicines, diagnosis, biosensors, bio-reactors and so on. The progress of gene technology has facilitated the discovery of these proteins and enabled us to produce a large amount of the proteins. The fundamental technology is a cDNA cloning technique.
Information on the amino acid sequence of a protein is encoded by mRNA. If the mRNA is converted to a corresponding DNA, i.e., a complementary DNA (cDNA), the cDNA can be used for determining the primary structure of the protein and producing a large amount of the protein. Therefore, various cDNA cloning techniques have been developed which include isolating mRNA from cells, synthesizing cDNAs from the mRNA, and isolating a cDNA encoding a target protein.
The most important requisite for cDNA cloning is to synthesize a cDNA containing an entire protein coding region encoded by an intact mRNA. The intact mRNA contains a characteristic structure at the 5' terminal, so called a cap. The cDNA containing an entire sequence involving from the nucleotide at which the cap is added is called a "full-length cDNA". If a full-length cDNA is synthesized, one can know the whole information on the primary structure of a protein and also use it for mass production of the protein encoded by the cDNA. According to conventional terminology, a "full-length cDNA" has been used as a cDNA containing an entire coding region of the protein even if the cDNA contains no cap site sequence. In this invention, however, a "full-length cDNA" is defined only as a cDNA containing a nucleotide sequence starting from the cap site.
The Gubler-Hoffman method [Gene 25:263-269(1983)], which is most widely used for cDNA synthesis, could not give a full-length cDNA, because deletion at the terminals of cDNA occurs. On the other hand, the Okayama-Berg method [Mol.Cell.Biol. 2:161-170(1982)] is known to give a full-length cDNA at high efficiency. The characteristic feature of this method is to add a dC tail to the 3' end of the first strand cDNA synthesized from mRNA. This method, however, does not always give a full-length cDNA, because dC tailing also occurs on truncated cDNA generated by unexpected termination of cDNA synthesis. The truncated cDNAs originated from the degraded mRNAs are also synthesized.
DISCLOSURE OF THE INVENTION
It is therefore an object of the present invention to provide a method for selectively synthesizing a full-length cDNA containing a nucleotide sequence starting from a cap site of mRNA, and a cDNA containing whole information on the primary structure of a protein. Furthermore, another object of this invention is to provide a method to prepare an intermediate for synthesizing the full-length cDNA and to prepare a recombinant vector containing the full-length cDNA.
The inventors succeeded in synthesizing a full-length cDNA at high efficiency from mRNA whose cap structure was replaced by a DNA oligonucleotide or a DNA-RNA chimeric oligonucleotide.
The invention provides a process for producing an intermediate for the synthesis of a full-length cDNA, which comprises a step of treating mRNA extracted from cells with an alkaline phosphatase to eliminate the phosphate group from the 5' end of an uncapped degraded mRNA, a step of decapping from the 5' end of a capped intact mRNA, and a step of ligating either a DNA oligonucleotide or a DNA-RNA chimeric oligonucleotide represented by the following general formula [I] to the 5' end phosphate group formed in the above step by the action of T4 RNA ligase, thereby selectively adding either the DNA oligonucleotide or the DNA-RNA chimeric oligonucleotide having an arbitrary sequence to the 5' end of the intact mRNA. dGMP and dTMP; N represents a ribonucleotide selected from among AMP, CMP, GMP and UMP; "-" represents a p
REFERENCES:
Hogrefe et al. (1990), J. Biol. Chem. 265(10):5561-5566.
Egli et al. (1992), Proc. Natl. Acad. Sci. USA 89:534-538.
Benevides et al. (1986), Biochemistry 25:41-50.
Edery et al. (1995), Mol. Cell. Biol. 15(6): 3363-3371.
Kato Seishi
Sekine Shingo
Carter Philip W.
Fleisher Mindy
Sagami Chemical Research Center
The Kanagawa Academy of Science and Technology Foundation
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