Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1986 Nov 11;14(21):8615–8624. doi: 10.1093/nar/14.21.8615

Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy.

H Haymerle, J Herz, G M Bressan, R Frank, K K Stanley
PMCID: PMC311881  PMID: 3024111

Abstract

We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E. coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.

Full text

PDF
8616

Images in this article

8619Image
on p.8619
8622Image
on p.8622

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bressan G. M., Castellani I., Colombatti A., Volpin D. Isolation and characterization of a 115,000-dalton matrix-associated glycoprotein from chick aorta. J Biol Chem. 1983 Nov 10;258(21):13262–13267. [PubMed] [Google Scholar]
  2. Bressan G. M., Castellani I., Giro M. G., Volpin D., Fornieri C., Pasquali Ronchetti I. Banded fibers in tropoelastin coacervates at physiological temperatures. J Ultrastruct Res. 1983 Mar;82(3):335–340. doi: 10.1016/s0022-5320(83)80021-5. [DOI] [PubMed] [Google Scholar]
  3. Grosjean H., Fiers W. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982 Jun;18(3):199–209. doi: 10.1016/0378-1119(82)90157-3. [DOI] [PubMed] [Google Scholar]
  4. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  5. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  6. Jay G., Khoury G., Seth A. K., Jay E. Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5543–5548. doi: 10.1073/pnas.78.9.5543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lathe R., Kieny M. P., Skory S., Lecocq J. P. Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA. 1984;3(2):173–182. doi: 10.1089/dna.1984.3.173. [DOI] [PubMed] [Google Scholar]
  8. Senapathy P. Origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2133–2137. doi: 10.1073/pnas.83.7.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Stanley K. K., Luzio J. P. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J. 1984 Jun;3(6):1429–1434. doi: 10.1002/j.1460-2075.1984.tb01988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Werner D., Chemla Y., Herzberg M. Isolation of poly(A)+ RNA by paper affinity chromatography. Anal Biochem. 1984 Sep;141(2):329–336. doi: 10.1016/0003-2697(84)90050-2. [DOI] [PubMed] [Google Scholar]
  11. Woods D. E., Markham A. F., Ricker A. T., Goldberger G., Colten H. R. Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5661–5665. doi: 10.1073/pnas.79.18.5661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES