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Proc Natl Acad Sci U S A. Mar 1, 1991; 88(5): 1943–1947.

Construction of a uniform-abundance (normalized) cDNA library.


We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.

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  • Bishop JO, Morton JG, Rosbash M, Richardson M. Three abundance classes in HeLa cell messenger RNA. Nature. 1974 Jul 19;250(463):199–204. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Li HH, Gyllensten UB, Cui XF, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. [PubMed]
  • Weissman SM. Molecular genetic techniques for mapping the human genome. Mol Biol Med. 1987 Jun;4(3):133–143. [PubMed]
  • Galau GA, Klein WH, Britten RJ, Davidson EH. Significance of rare m RNA sequences in liver. Arch Biochem Biophys. 1977 Mar;179(2):584–599. [PubMed]
  • Britten RJ, Graham DE, Neufeld BR. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. [PubMed]
  • Smith MJ, Britten RJ, Davidson EH. Studies on nucleic acid reassociation kinetics: reactivity of single-stranded tails in DNA-DNA renaturation. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4805–4809. [PMC free article] [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martial JA, Baxter JD. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. [PubMed]
  • Gubler U, Hoffman BJ. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. [PubMed]
  • Ogden RC, Adams DA. Electrophoresis in agarose and acrylamide gels. Methods Enzymol. 1987;152:61–87. [PubMed]
  • Houck CM, Rinehart FP, Schmid CW. A ubiquitous family of repeated DNA sequences in the human genome. J Mol Biol. 1979 Aug 15;132(3):289–306. [PubMed]
  • Wilson GN, Hollar BA, Waterson JR, Schmickel RD. Molecular analysis of cloned human 18S ribosomal DNA segments. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5367–5371. [PMC free article] [PubMed]
  • Norment AM, Littman DR. A second subunit of CD8 is expressed in human T cells. EMBO J. 1988 Nov;7(11):3433–3439. [PMC free article] [PubMed]
  • Maddon PJ, Littman DR, Godfrey M, Maddon DE, Chess L, Axel R. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell. 1985 Aug;42(1):93–104. [PubMed]
  • Lefranc MP, Rabbitts TH. Two tandemly organized human genes encoding the T-cell gamma constant-region sequences show multiple rearrangement in different T-cell types. Nature. 1985 Aug 1;316(6027):464–466. [PubMed]
  • Gunning P, Ponte P, Okayama H, Engel J, Blau H, Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol. 1983 May;3(5):787–795. [PMC free article] [PubMed]
  • Chorney MJ, Sawada I, Gillespie GA, Srivastava R, Pan J, Weissman SM. Transcription analysis, physical mapping, and molecular characterization of a nonclassical human leukocyte antigen class I gene. Mol Cell Biol. 1990 Jan;10(1):243–253. [PMC free article] [PubMed]
  • He X, Treacy MN, Simmons DM, Ingraham HA, Swanson LW, Rosenfeld MG. Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature. 1989 Jul 6;340(6228):35–41. [PubMed]
  • Wilson JT, Wilson LB, deRiel JK, Villa-komaroff L, Efstratiadis A, Forget BG, Weissman SM. Insertion of synthetic copies of human globin genes into bacterial plasmids. Nucleic Acids Res. 1978 Feb;5(2):563–581. [PMC free article] [PubMed]
  • Moon RT, McMahon AP. Generation of diversity in nonerythroid spectrins. Multiple polypeptides are predicted by sequence analysis of cDNAs encompassing the coding region of human nonerythroid alpha-spectrin. J Biol Chem. 1990 Mar 15;265(8):4427–4433. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. [PMC free article] [PubMed]
  • Stafford DW, Bieber D. Concentration of DNA solutions by extraction with 2-butanol. Biochim Biophys Acta. 1975 Jan 6;378(1):18–21. [PubMed]
  • Singer MF. Highly repeated sequences in mammalian genomes. Int Rev Cytol. 1982;76:67–112. [PubMed]
  • Ko MS. An 'equalized cDNA library' by the reassociation of short double-stranded cDNAs. Nucleic Acids Res. 1990 Oct 11;18(19):5705–5711. [PMC free article] [PubMed]
  • Vasavada HA, Ganguly S, Settleman J, Dimaio D, Weissman S. A model system for the rescue of cDNA encoding transacting transcription activator by contingent replication assay. Indian J Biochem Biophys. 1988 Dec;25(6):488–494. [PubMed]
  • Kandpal RP, Ward DC, Weissman SM. Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping. Nucleic Acids Res. 1990 Apr 11;18(7):1789–1795. [PMC free article] [PubMed]
  • Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. [PubMed]

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