Abstract
Molecular cloning is the process of inserting foreign fragments of DNA into a plasmid or bacteriophage vector that is capable of autonomous replication in a suitable host cell. The resulting recombinant DNA molecules can then be amplified by growth in the host and isolated in pure form. The nucleotide sequence of the inserted portion of the recombinant molecule can shed light on the structure of a particular gene or messenger RNA (mRNA) and provide the primary amino acid sequence of the protein it encodes. Because of the greater accuracy and rapidity of nucleotide sequence analysis over conventional protein sequence analysis, this is now the standard method for elucidating the primary structure of a protein. Recombinant clones may be used to generate probes for monitoring mRNA expression either by Northern blotting, RNase protection, or in situ hybridization, and, thus, the developmental and anatomical distribution of the mRNA may be determined. Recombinant clones can be tailored to produce large amounts of the encoded protein in bacterial or mammalian tissue culture systems. The cloned DNA can be altered by any of a number of in vitro mutagenesis procedures, thus altering the amino acid sequence of the encoded protein.
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References
Aviv H. and Leder R. (1972) Purification of biologically active globin messenger RNA by chromography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408–1412.
Chaudhan N., Hahn W. E. (1983) Genetic expression in the developing brain. Science 220, 924–928.
Chirgwin J. M., Przybyla A. E., MacDonald R. J., and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18, 5294–5299.
Chikaraishi D. M. (1979) Complexity of cytoplasmic polyadenylated and nonpolyadenylated rat brain ribonucleic acid. Biochemistry 18, 3249–3256
Dworkin M. B and Dawid I. B. (1980) Use of a cloned library for the study of abundant poly(A)+ RNA during Xenopus laevis development. Dev Biol. 76, 449–464.
Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 132, 6–13
Gergen J. P, Stern R. H., and Wensink P. C. (1979) Filter replicas and permanent collections of recombinant DNA plasmids, Nucl Acids Res. 7, 2115–2136
Grunstein M. and Wallis J. (1979) Methods in Enzymology (Wu R., ed.), vol. 68, pp 379–389 Academic Press, New York.
Hedrick S. M., Cohen D. I, Nielsen E. A., and Davis M. M. (1984) Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature 308, 149–153.
Hood L. E., Wilson J H., and Wood W. B. (1975) Molecular Biology of Eucaryotic Cells. A Problems Approach, vol. 1, The Benjamin/Cummings Publishing Company, Menlo Park, California.
Kaufman D. L., McGinnis J. F., Kneger N. R., and Tobin A. J. (1986) Brain glutamate decarboxylase cloned in λgt-11 Fusion protein produces δ-aminobutyric acid. Science 232, 1138–1140.
Kohne D. E., Levison S. A., and Byers M. J. (1977) Room temperature method for increasing the rate of DNA reassociation by many thousandfold: The phenol emulsion reassociation technique. Biochemistry 16, 5329–5341.
Maniatis T., Fntsch E. G., and Sambrook J. (1982) Molecular Cloning. Cold Spring Harbor, New York: Cold Spring Harbor Laboratories.
Masu Y., Nakayama K., Tamaki H., Harada Y., Kuno M., and Nakanishi S. (1987) cDNA cloning of bovine substance-K receptor through oocyte expression system. Nature 329, 836–838
Meyuhas O. and Perry R. P. (1979) Relationship between size, stability, and abundance of the messenger RNA or mouse L cells. Cell 16, 139–148.
Miller F. D., Naus C. C. G., Higgins G. A., Bloom F. E., and Milner R. J. (1987) Developmentally regulated rat brain mRNAs. Molecular and anatomical characterization. J Neurosci. 7, 2433–2444.
Milner R. J. and Sutcliffe J. G. (1983) Gene expression in rat brain Nucl Acids Res. 11, 5497–5520.
Milner R. J., Lai C., Nave K-A., Lenoir D., Ogata J., and Sutcliffe J. G. (1985) Nucleotide sequences of two mRNAs for rat bram myelin proteohpid protein. Cell 42, 931–939.
Milner R. J., Bloom F E., and Sutcliffe J G. (1987) Brain specific genes: Strategies and issues. Current Topics in Developmental Biology 21, 117–150.
Nathans J., Thomas D., and Hogness D. S. (1986) Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232, 193–202.
Ogata R. T., Shreffler D. C, Sepich D. S., and Lilly S. P. (1983) cDNA clone spanning the α-δ subunit junction in the precursor of the murine fourth complement component (C4). Proc. Natl Acad. Sci. USA 80, 5061–5065.
Okayama H. and Berg P. (1983) A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell. Biol. 3, 280–289.
Schibler K, Tosi M., Pittet A.-C, Fabiani L., and Wellauer P. K. (1980) Tissue-specific expression of mouse α-amylase genes. J. Mol. Biol. 142, 93–116.
Sikela J. M. and Hahn W. E. (1987) Screening an expression library with a ligand probe: Isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein Proc. Natl Acad. Set USA 84, 3038–3042.
Sutcliffe J. G. (1988) mRNA in the mammalian central nervous system. Ann. Rev. Neurosct. 11, 157–198.
Timberlake W E. (1980) Developmental gene regulation in Aspergillus nidulans. Dev. Biol. 78, 497–510.
Travis G. H., Naus C. G., Morrison J. H., Bloom F. E., and Sutcliffe J. G. (1987) Subtractive cloning of complementary DNAs and analysis of messenger RNAs with regional heterogeneous distributions in primate cortex. Neuropharmacology 26, 845–854
Travis G. H. and Sutcliffe J. G. (1988) Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: Isolation of low abundance monkey cortex-specific mRNAs. Proc. Natl. Acad. Sa. USA 85, 1696–1700.
Wietgrefe S., Zupancic M., Haase A., Chesebro B., Race R., Frey II W., Rustan T., and Friedman R. L. (1985) Cloning of a gene whose expression is increased in scrapie and m senile plaques in human brain. Science 230, 1177–1179.
Young R. A. and Davis R. W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl Acad. Set. USA 80, 1194–1198.
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Travis, G.H., Milner, R.J., Sutcliffe, J.G. (1990). Preparation and Use of Subtractive cDNA Hybridization Probes for cDNA Cloning. In: Boulton, A.A., Baker, G.B., Campagnoni, A.T. (eds) Molecular Neurobiological Techniques. Neuromethods, vol 16. Humana Press. https://doi.org/10.1385/0-89603-140-3:49
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DOI: https://doi.org/10.1385/0-89603-140-3:49
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