Method for generating a subtracted cDNA library and uses of the

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

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435 6, 435 912, 435 691, 4351723, 435810, 436501, 536 231, 536 241, 536 243, 536 2431, 536 2432, 536 2433, 935 9, 935 77, 935 78, C12P 1934

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056437617

ABSTRACT:
This invention provides a method of generating a subtracted cDNA library of a cell comprising: a) generating a cDNA library of the cell; b) isolating double-stranded DNAs from the cDNA library; c) releasing the double-stranded cDNA inserts from the double-stranded DNAs; d) denaturing the isolated double-stranded cDNA inserts; e) hybridizing the denatured double-stranded cDNA inserts with a labelled single-stranded nucleic acid molecules which are to be subtracted from the cDNA library; and f) separating the hybridized labeled single-stranded nucleic acid molecule from the double-stranded cDNA inserts, thereby generating a subtracted cDNA library of a cell.

REFERENCES:
Duguid, J.R., et al. (1988) Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library, Proc. Natl. Acad. Sci., USA, 85:5738-5742 (Exhibit B).
Hara, E., et al. (1991) Subtractive cDNA cloning using oligo(dT)-30-latex and PCR: Isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells, Nucleic Acids Research, 19(25):7097-7104.
Herfort, M.R., et al. (Nov. 1991) Simple and efficient subtractive hybridization screening, BioTechniques, 11(5):598-603 (Exhibit D).
Lee, S.W., et al. (Apr. 1991) Positive selection of candidate tumor-suppressor genes by subtractive hybridization, Proc. Natl. Acad. Sci, USA, 88:2825-2829 (Exhibit E).
Maniatis, T., et al. (1982) Strategies for cDNA cloning, Molecular Cloning, A Laboratory Manual, pp. 224-228 (Exhibit F).
Sive, H.L., et al. (Nov. 1988) A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction, Nucleic Acids Research, 16(22)10937 (Exhibit G).
Travis, G.H., et al. (Mar. 1988) Phenol emulsion-enhanced DNA driven subtractive cDNA cloning: Isolation of low-abundance monkey cortex-specific mRNAs, Proc. Natl. Acad. Sci. USA, 85:1696-1700 (Exhibit H).
Wieland, I., et al. (1990) A method for difference cloning, gene amplification following subtractive hybridization, Proc. Natl. Acad. Sci., USA, 87:2720-2724 (Exhibit I).
Audette et al. (1989) Molecular Immunology, vol. 26, No. 6, pp. 515-522.
Hara et al. (1993) Analytical Biochemistry, vol. 214, pp. 58-64.
Rubenstein et al. (1990) Nuc. Acid Res, vol. 18, No. 17, pp. 4833-4842.

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