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. 1975 Jan;2(1):43–60. doi: 10.1093/nar/2.1.43

Enzymatic multiplication of a chemically synthesized DNA fragment.

K Olson, T Gabriel, J Michalewsky, C Harvey
PMCID: PMC342810  PMID: 1093143

Abstract

A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2', 3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques. All template strands were copied with complete repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.

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Selected References

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

  1. Atkinson M. R., Deutscher M. P., Kornberg A., Russell A. F., Moffatt J. G. Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2',3'-dideoxyribonucleotide. Biochemistry. 1969 Dec;8(12):4897–4904. doi: 10.1021/bi00840a037. [DOI] [PubMed] [Google Scholar]
  2. Brutlag D., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3' leads to 5' exonuclease activity in deoxyribonucleic acid polymerases. J Biol Chem. 1972 Jan 10;247(1):241–248. [PubMed] [Google Scholar]
  3. Chang L. M., Bollum F. J. Deoxynucleotide-polymerizing enzymes of calf thymus gland. V. Homogeneous terminal deoxynucleotidyl transferase. J Biol Chem. 1971 Feb 25;246(4):909–916. [PubMed] [Google Scholar]
  4. Cook A. F., de Czekala A., Gabriel T. F., Harvey C. L., Holman M., Michaelewsky J. E., Nussbaum A. L. High pressure liquid chromatography of oligodeoxyribonucleotides. Biochim Biophys Acta. 1973 Oct 26;324(3):433–439. doi: 10.1016/0005-2787(73)90288-8. [DOI] [PubMed] [Google Scholar]
  5. Gefter M. L., Molineux I. J., Kornberg T., Khorana H. G. Deoxyribonucleic acid synthesis in cell-free extracts. 3. Catalytic properties of deoxyribonucleic acid polymerase II. J Biol Chem. 1972 May 25;247(10):3321–3326. [PubMed] [Google Scholar]
  6. Harvey C. L., Wright R., Cook A. F., Maichuk D. T., Nussbaum A. L. Use of phosphate-blocking groups in ligase joining oligodeoxyribonucleotides. Biochemistry. 1973 Jan 16;12(2):208–214. doi: 10.1021/bi00726a005. [DOI] [PubMed] [Google Scholar]
  7. Harvey C. L., Wright R. Ligase joining of oligodeoxythymidylates. Biochemistry. 1972 Jul 4;11(14):2667–2671. doi: 10.1021/bi00764a018. [DOI] [PubMed] [Google Scholar]
  8. Jovin T. M., Englund P. T., Bertsch L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):2996–3008. [PubMed] [Google Scholar]
  9. Kleppe K., Ohtsuka E., Kleppe R., Molineux I., Khorana H. G. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases. J Mol Biol. 1971 Mar 14;56(2):341–361. doi: 10.1016/0022-2836(71)90469-4. [DOI] [PubMed] [Google Scholar]
  10. LEHMAN I. R., RICHARDSON C. C. THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. IV. AN EXONUCLEASE ACTIVITY PRESENT IN PURIFIED PREPARATIONS OF DEOXYRIBONUCLEIC ACID POLYMERASE. J Biol Chem. 1964 Jan;239:233–241. [PubMed] [Google Scholar]
  11. Masamune Y., Fleischman R. A., Richardson C. C. Enzymatic removal and replacement of nucleotides at single strand breaks in deoxyribonucleic acid. J Biol Chem. 1971 Apr 25;246(8):2680–2691. [PubMed] [Google Scholar]
  12. Padmanabhan R., Wu R. Nucleotide sequence analysis of DNA. IX. Use of oligonucleotides of defined sequence as primers in DNA sequence analysis. Biochem Biophys Res Commun. 1972 Sep 5;48(5):1295–1302. doi: 10.1016/0006-291x(72)90852-2. [DOI] [PubMed] [Google Scholar]
  13. Panet A., Khorana H. G. Studies on polynucleotides. The linkage of deoxyribopolynucleotide templates to cellulose and its use in their replication. J Biol Chem. 1974 Aug 25;249(16):5213–5221. [PubMed] [Google Scholar]
  14. Wickner W., Schekman R., Geider K., Kornberg A. A new form of DNA polymerase 3 and a copolymerase replicate a long, single-stranded primer-template. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1764–1767. doi: 10.1073/pnas.70.6.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]

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