Home LiteratureArticle Details
PMID: 7937745 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Construction and characterization of a normalized cDNA library.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 91 ·No. 20 ·1994-09-27 ·Pages 9228-32

Soares MB, Bonaldo MF, Jelene P, Su L, Lawton L, Efstratiadis A

Abstract

We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

MeSH Terms
Base Sequence Brain/metabolism Cloning, Molecular DNA Primers DNA Probes DNA, Complementary Female Gene Library Genetic Vectors Humans Infant Molecular Sequence Data RNA, Messenger/metabolism Spinal Muscular Atrophies of Childhood/genetics,metabolism
Chemicals
DNA Primers DNA Probes DNA, Complementary RNA, Messenger
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Soares M B
Department of Psychiatry, Columbia University, New York, NY 10032.
Bonaldo M F
Jelene P
Su L
Lawton L
Efstratiadis A
References (30)
30 references, click to expand
  1. A survey on intron and exon lengths.
    Nucleic Acids Res. 1988 Nov 11;16(21):9893-908 PMID: 3057449
  2. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system.
    Nucleic Acids Res. 1994 Mar 25;22(6):987-92 PMID: 8152931
  3. Subtractive hybridization system using single-stranded phagemids with directional inserts.
    Nucleic Acids Res. 1990 Aug 25;18(16):4833-42 PMID: 2168539
  4. An 'equalized cDNA library' by the reassociation of short double-stranded cDNAs.
    Nucleic Acids Res. 1990 Oct 11;18(19):5705-11 PMID: 2216762
  5. Basic local alignment search tool.
    J Mol Biol. 1990 Oct 5;215(3):403-10 PMID: 2231712
  6. Single pass sequencing and physical and genetic mapping of human brain cDNAs.
    Nat Genet. 1992 Nov;2(3):180-5 PMID: 1345165
  7. Changes in nucleic acid and protein content of the human brain during growth.
    Pediatr Res. 1968 Sep;2(5):352-5 PMID: 5672695
  8. Three abundance classes in HeLa cell messenger RNA.
    Nature. 1974 Jul 19;250(463):199-204 PMID: 4855195
  9. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.
    Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961-5 PMID: 1105573
  10. Complexity and characterization of polyadenylated RNA in the mouse brain.
    Cell. 1976 May;8(1):139-50 PMID: 986249
  11. Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.
    Proc Natl Acad Sci U S A. 1977 Mar;74(3):1020-3 PMID: 265546
  12. A program for least squares analysis of reassociation and hybridization data.
    Nucleic Acids Res. 1977 Jun;4(6):1727-37 PMID: 896473
  13. Sequence diversity studies of rat brain RNA: effects of environmental complexity on rat brain RNA diversity.
    J Neurochem. 1978 Jan;30(1):191-203 PMID: 621507
  14. Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.
    Proc Natl Acad Sci U S A. 1978 Nov;75(11):5544-7 PMID: 281703
  15. Regulation of gene expression: possible role of repetitive sequences.
    Science. 1979 Jun 8;204(4397):1052-9 PMID: 451548
  16. Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics.
    Nucleic Acids Res. 1982 Dec 20;10(24):8061-77 PMID: 6186985
  17. pEMBL: a new family of single stranded plasmids.
    Nucleic Acids Res. 1983 Mar 25;11(6):1645-55 PMID: 6300771
  18. Genetic expression in the developing brain.
    Science. 1983 May 27;220(4600):924-8 PMID: 6189184
  19. Gene expression in rat brain.
    Nucleic Acids Res. 1983 Aug 25;11(16):5497-520 PMID: 6193485
  20. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
    Anal Biochem. 1983 Jul 1;132(1):6-13 PMID: 6312838
  21. Discrete poly(A)- RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5'-termini.
    Mol Biol Rep. 1983 Aug;9(3):155-61 PMID: 6195519
  22. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum.
    Anal Biochem. 1984 Feb;137(1):266-7 PMID: 6329026
  23. Polyadenylation of a human mitochondrial ribosomal RNA transcript detected by molecular cloning.
    Gene. 1985;35(3):305-12 PMID: 4043734
  24. Molecular genetic techniques for mapping the human genome.
    Mol Biol Med. 1987 Jun;4(3):133-43 PMID: 3306263
  25. Production of single-stranded plasmid DNA.
    Methods Enzymol. 1987;153:3-11 PMID: 3323803
  26. A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro.
    Mol Cell Biol. 1988 Oct;8(10):4502-9 PMID: 3185559
  27. Construction of a uniform-abundance (normalized) cDNA library.
    Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1943-7 PMID: 1705712
  28. Brain non-adenylated mRNAs.
    Brain Res Brain Res Rev. 1992 Sep-Dec;17(3):263-82 PMID: 1361395
  29. Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.
    Nat Genet. 1993 Aug;4(4):373-80 PMID: 8401585
  30. Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.
    Genet Anal Tech Appl. 1990 Feb;7(1):5-17 PMID: 1691011
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.91.20.9228
Published
1994-09-27
Pages
9228-32
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC44785
Subset
IM
Grants
NICHD NIH HHS · 1R55HD28422 · United States
NCRR NIH HHS · 2 S07 RR05395-03 · United States
NHGRI NIH HHS · HG00362 · United States
External Links
PubMed source DOI: 10.1073/pnas.91.20.9228 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2