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. 1974 Jun;71(6):2510–2514. doi: 10.1073/pnas.71.6.2510

Chemical Synthesis of a Primer and Its Use in the Sequence Analysis of the Lysozyme Gene of Bacteriophage T4*

R Padmanabhan 1,†, Ernest Jay 1, Ray Wu 1
PMCID: PMC388489  PMID: 4526223

Abstract

We have developed a general approach for determining the nucleotide sequence of a gene, with the aid of a deoxyribonucleotide primer of defined sequence. The selection of the primer sequence was based on a short segment of mRNA sequence of T4 phage lysozyme. A tetradecadeoxyribonucleotide primer was chemically synthesized and its sequence verified by sequence analysis. This primer was found to bind to the single-stranded region of the exonuclease III-treated T4 DNA, and specific nucleotides were incorporated to its 3′ end. The result indicated that this primer was bound to the expected location on the T4 DNA. Therefore, long sequences of the T4 lysozyme gene can now be determined from this specific starting point.

Keywords: DNA sequencing, repair synthesis, hybridization

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Selected References

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

  1. Agarwal K. L., Yamazaki A., Khorana H. G. Studies on polynucleotides. 98. A convenient and general method for the preparation of protected dideoxyribonucleotides containing 5'-phosphate end groups. J Am Chem Soc. 1971 Jun 2;93(11):2754–2762. doi: 10.1021/ja00740a028. [DOI] [PubMed] [Google Scholar]
  2. Bernardi A., Bernardi G. Studies on acid hydrolases. IV. Isolation and characterization of spleen exonuclease. Biochim Biophys Acta. 1968 Feb 26;155(2):360–370. [PubMed] [Google Scholar]
  3. Brownlee G. G., Sanger F. Chromatography of 32P-labelled oligonucleotides on thin layers of DEAE-cellulose. Eur J Biochem. 1969 Dec;11(2):395–399. doi: 10.1111/j.1432-1033.1969.tb00786.x. [DOI] [PubMed] [Google Scholar]
  4. Caruthers M. H., Khorana H. G. CXI. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Synthesis of a dodecadeoxynucleotide and a heptadeoxynucleotide corresponding to the nucleotide sequence 66 to 77. J Mol Biol. 1972 Dec 28;72(2):407–426. doi: 10.1016/0022-2836(72)90154-4. [DOI] [PubMed] [Google Scholar]
  5. Doel M. T., Smith M. The chemical synthesis of deoxyribo-oligonucleotides complementary to a portion of the lysozyme gene of phage T4 and their hybridization to phage specific RNA and phage DNA. FEBS Lett. 1973 Aug 1;34(1):99–102. doi: 10.1016/0014-5793(73)80712-4. [DOI] [PubMed] [Google Scholar]
  6. Ghangas G. S., Jay E., Bambara R., Wu R. Nucleotide sequence analysis of DNA. XI. The 3' terminal sequences of bacteriophage lambda and phi 80 DNA. Biochem Biophys Res Commun. 1973 Oct 1;54(3):998–1007. doi: 10.1016/0006-291x(73)90793-6. [DOI] [PubMed] [Google Scholar]
  7. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [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. Kasai T., Bautz E. K., Guha A., Szybalski W. Identification of the transcribing DNA strand for the rII and endolysin genes of coliphage T4. J Mol Biol. 1968 Jun 28;34(3):709–712. doi: 10.1016/0022-2836(68)90192-7. [DOI] [PubMed] [Google Scholar]
  10. Loewen P. C., Khorana H. G. Studies on polynucleotides. CXXII. The dodecanucleotide sequence adjoining the C-C-A end of the tyrosine transfer ribonucleic acid gene. J Biol Chem. 1973 May 25;248(10):3489–3499. [PubMed] [Google Scholar]
  11. NEU H. C., HEPPEL L. A. NUCLEOTIDE SEQUENCE ANALYSIS OF POLYRIBONUCLEOTIDES BY MEANS OF PERIODATE OXIDATION FOLLOWED BY CLEAVAGE WITH AN AMINE. J Biol Chem. 1964 Sep;239:2927–2934. [PubMed] [Google Scholar]
  12. Narang S. A., Itakura K., Bahl C. P., Wigfield Y. Y. Chemical synthesis of two deoxyribopolynucleotide fragments containing the natural sequence of T4 lysozyme gene e. Biochem Biophys Res Commun. 1972 Oct 17;49(2):445–452. doi: 10.1016/0006-291x(72)90431-7. [DOI] [PubMed] [Google Scholar]
  13. Nirenberg M., Leder P., Bernfield M., Brimacombe R., Trupin J., Rottman F., O'Neal C. RNA codewords and protein synthesis, VII. On the general nature of the RNA code. Proc Natl Acad Sci U S A. 1965 May;53(5):1161–1168. doi: 10.1073/pnas.53.5.1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roychoudhury R., Fischer D., Kössel H. A new method for the sequence analysis of oligodeoxynucleotides. Biochem Biophys Res Commun. 1971 Oct 15;45(2):430–435. doi: 10.1016/0006-291x(71)90837-0. [DOI] [PubMed] [Google Scholar]
  17. STREISINGER G., EDGAR R. S., DENHARDT G. H. CHROMOSOME STRUCTURE IN PHAGE T4. I. CIRCULARITY OF THE LINKAGE MAP. Proc Natl Acad Sci U S A. 1964 May;51:775–779. doi: 10.1073/pnas.51.5.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sanger F., Donelson J. E., Coulson A. R., Kössel H., Fischer D. Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage fl DNA. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1209–1213. doi: 10.1073/pnas.70.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Southern E. M., Mitchell A. R. Chromatography of nucleic acid digests on thin layers of cellulose impregnated with polyethyleneimine. Biochem J. 1971 Jul;123(4):613–617. doi: 10.1042/bj1230613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Streisinger G., Okada Y., Emrich J., Newton J., Tsugita A., Terzaghi E., Inouye M. Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol. 1966;31:77–84. doi: 10.1101/sqb.1966.031.01.014. [DOI] [PubMed] [Google Scholar]
  21. THOMAS C. A., Jr, MACHATTIE L. A. CIRCULAR T2 DNA MOLECULES. Proc Natl Acad Sci U S A. 1964 Nov;52:1297–1301. doi: 10.1073/pnas.52.5.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tsugita A., Inouye M. Complete primary structure of phage lysozyme from Escherichia coli T4. J Mol Biol. 1968 Oct 14;37(1):201–212. doi: 10.1016/0022-2836(68)90083-1. [DOI] [PubMed] [Google Scholar]
  23. Wu R. Nucleotide sequence analysis of DNA. I. Partial sequence of the cohesive ends of bacteriophage lambda and 186 DNA. J Mol Biol. 1970 Aug;51(3):501–521. doi: 10.1016/0022-2836(70)90004-5. [DOI] [PubMed] [Google Scholar]
  24. Wu R. Nucleotide sequence analysis of DNA. Nat New Biol. 1972 Apr 19;236(68):198–200. doi: 10.1038/newbio236198a0. [DOI] [PubMed] [Google Scholar]
  25. Wu R., Taylor E. Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA. J Mol Biol. 1971 May 14;57(3):491–511. doi: 10.1016/0022-2836(71)90105-7. [DOI] [PubMed] [Google Scholar]
  26. Wu R., Tu C. D., Padmanabhan R. Nucleotide sequence analysis of DNA. XII. The chemical synthesis and sequence analysis of a dodecadeoxynucleotide which binds to the endolysin gene of bacteriophage lambda. Biochem Biophys Res Commun. 1973 Dec 19;55(4):1092–1099. doi: 10.1016/s0006-291x(73)80007-5. [DOI] [PubMed] [Google Scholar]

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