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. 1978 Apr;75(4):1763–1767. doi: 10.1073/pnas.75.4.1763

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

S P Goff, P Berg
PMCID: PMC392420  PMID: 347445

Abstract

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

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Selected References

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

  1. Chase J. W., Richardson C. C. Exonuclease VII of Escherichia coli. Mechanism of action. J Biol Chem. 1974 Jul 25;249(14):4553–4561. [PubMed] [Google Scholar]
  2. Chase J. W., Richardson C. C. Exonuclease VII of Escherichia coli. Purification and properties. J Biol Chem. 1974 Jul 25;249(14):4545–4552. [PubMed] [Google Scholar]
  3. Clarke L., Carbon J. Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4361–4365. doi: 10.1073/pnas.72.11.4361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen S. N., Chang A. C., Boyer H. W., Helling R. B. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240–3244. doi: 10.1073/pnas.70.11.3240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  6. Glover D. M., White R. L., Finnegan D. J., Hogness D. S. Characterization of six cloned DNAs from Drosophila melanogaster, including one that contains the genes for rRNA. Cell. 1975 Jun;5(2):149–157. doi: 10.1016/0092-8674(75)90023-9. [DOI] [PubMed] [Google Scholar]
  7. Goff S. P., Berg P. Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells. Cell. 1976 Dec;9(4 Pt 2):695–705. doi: 10.1016/0092-8674(76)90133-1. [DOI] [PubMed] [Google Scholar]
  8. Heyneker H. L., Shine J., Goodman H. M., Boyer H. W., Rosenberg J., Dickerson R. E., Narang S. A., Itakura K., Lin S., Riggs A. D. Synthetic lac operator DNA is functional in vivo. Nature. 1976 Oct 28;263(5580):748–752. doi: 10.1038/263748a0. [DOI] [PubMed] [Google Scholar]
  9. Hofstetter H., Schamböck A., Van Den Berg J., Weissmann C. Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method. Biochim Biophys Acta. 1976 Dec 13;454(3):587–591. doi: 10.1016/0005-2787(76)90286-0. [DOI] [PubMed] [Google Scholar]
  10. Jackson D. A., Symons R. H., Berg P. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2904–2909. doi: 10.1073/pnas.69.10.2904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lobban P. E., Kaiser A. D. Enzymatic end-to end joining of DNA molecules. J Mol Biol. 1973 Aug 15;78(3):453–471. doi: 10.1016/0022-2836(73)90468-3. [DOI] [PubMed] [Google Scholar]
  13. Maniatis T., Kee S. G., Efstratiadis A., Kafatos F. C. Amplification and characterization of a beta-globin gene synthesized in vitro. Cell. 1976 Jun;8(2):163–182. doi: 10.1016/0092-8674(76)90001-5. [DOI] [PubMed] [Google Scholar]
  14. Marians K. J., Wu R., Stawinski J., Hozumi T., Narang S. A. Cloned synthetic lac operator DNA is biologically active. Nature. 1976 Oct 28;263(5580):744–748. doi: 10.1038/263744a0. [DOI] [PubMed] [Google Scholar]
  15. Morrow J. F., Cohen S. N., Chang A. C., Boyer H. W., Goodman H. M., Helling R. B. Replication and transcription of eukaryotic DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1974 May;71(5):1743–1747. doi: 10.1073/pnas.71.5.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  17. Rougeon F., Kourilsky P., Mach B. Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid. Nucleic Acids Res. 1975 Dec;2(12):2365–2378. doi: 10.1093/nar/2.12.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Shenk T. E., Carbon J., Berg P. Construction and analysis of viable deletion mutants of simian virus 40. J Virol. 1976 May;18(2):664–671. doi: 10.1128/jvi.18.2.664-671.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sogaramella V., Khorana H. G. Studies on polynucleotides. CXVI. A further study of the T4 ligase-catalyzed joining of DNA at base-paired ends. J Mol Biol. 1972 Dec 30;72(3):493–502. doi: 10.1016/0022-2836(72)90170-2. [DOI] [PubMed] [Google Scholar]
  20. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  21. Thomas M., Cameron J. R., Davis R. W. Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4579–4583. doi: 10.1073/pnas.71.11.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  23. Wensink P. C., Finnegan D. J., Donelson J. E., Hogness D. S. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. doi: 10.1016/0092-8674(74)90045-2. [DOI] [PubMed] [Google Scholar]

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