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. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760

Direct chemical method for sequencing RNA.

D A Peattie
PMCID: PMC383470  PMID: 377283

Abstract

Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P. After a partial, specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on a polyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.

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Selected References

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  1. Baralle F. E. Complete nucleotide sequence of the 5' noncoding region of rabbit beta-globin mRNA. Cell. 1977 Apr;10(4):549–558. doi: 10.1016/0092-8674(77)90088-5. [DOI] [PubMed] [Google Scholar]
  2. Brookes P., Lawley P. D. The reaction of mono- and di-functional alkylating agents with nucleic acids. Biochem J. 1961 Sep;80(3):496–503. doi: 10.1042/bj0800496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown N. L., Smith M. The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B. J Mol Biol. 1977 Oct 15;116(1):1–28. doi: 10.1016/0022-2836(77)90115-2. [DOI] [PubMed] [Google Scholar]
  4. Brownlee G. G., Cartwright E. M. Rapid gel sequencing of RNA by primed synthesis with reverse transcriptase. J Mol Biol. 1977 Jul;114(1):93–117. doi: 10.1016/0022-2836(77)90285-6. [DOI] [PubMed] [Google Scholar]
  5. Bruce A. G., Uhlenbeck O. C. Reactions at the termini of tRNA with T4 RNA ligase. Nucleic Acids Res. 1978 Oct;5(10):3665–3677. doi: 10.1093/nar/5.10.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cashmore A. R., Petersen G. B. The degradation of DNA by hydrazine: identification of 3-ureidopyrazole as a product of the hydrazinolysis of deoxycytidylic acid residues. Nucleic Acids Res. 1978 Jul;5(7):2485–2491. doi: 10.1093/nar/5.7.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang J. C., Temple G. F., Poon R., Neumann K. H., Kan Y. W. The nucleotide sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5145–5149. doi: 10.1073/pnas.74.11.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ehrenberg L., Fedorcsak I., Solymosy F. Diethyl pyrocarbonate in nucleic acid research. Prog Nucleic Acid Res Mol Biol. 1976;16:189–262. doi: 10.1016/s0079-6603(08)60758-8. [DOI] [PubMed] [Google Scholar]
  10. Hayes D. H., Hayes-Baron F. Hydrazinolysis of some purines and pyrimidines and their related nucleosides and nucleotides. J Chem Soc Perkin 1. 1967;16:1528–1533. doi: 10.1039/j39670001528. [DOI] [PubMed] [Google Scholar]
  11. Kramer F. R., Mills D. R. RNA sequencing with radioactive chain-terminating ribonucleotides. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5334–5338. doi: 10.1073/pnas.75.11.5334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lockard R. E., Alzner-Deweerd B., Heckman J. E., MacGee J., Tabor M. W., RajBhandary U. L. Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis. Nucleic Acids Res. 1978 Jan;5(1):37–56. doi: 10.1093/nar/5.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McGeoch D. J., Turnbull N. T. Analysis of the 3'-terminal nucleotide sequence of vesicular stomatitis virus N protein mRNA. Nucleic Acids Res. 1978 Nov;5(11):4007–4024. doi: 10.1093/nar/5.11.4007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miyazaki M. Studies on the nucleotide sequence of pseudouridine-containing 5S RNA from Saccharomyces cerevisiae. J Biochem. 1974 Jun;75(6):1407–1410. doi: 10.1093/oxfordjournals.jbchem.a130532. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Ross A., Brimacombe R. Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli. Nucleic Acids Res. 1978 Jan;5(1):241–256. doi: 10.1093/nar/5.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rubin G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 1975;12:45–64. doi: 10.1016/s0091-679x(08)60951-6. [DOI] [PubMed] [Google Scholar]
  19. Rubin G. M. The nucleotide sequence of Saccharomyces cerevisiae 5.8 S ribosomal ribonucleic acid. J Biol Chem. 1973 Jun 10;248(11):3860–3875. [PubMed] [Google Scholar]
  20. Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  21. Simoncsits A., Brownlee G. G., Brown R. S., Rubin J. R., Guilley H. New rapid gel sequencing method for RNA. Nature. 1977 Oct 27;269(5631):833–836. doi: 10.1038/269833a0. [DOI] [PubMed] [Google Scholar]
  22. Stanley J., Vassilenko S. A different approach to RNA sequencing. Nature. 1978 Jul 6;274(5666):87–89. doi: 10.1038/274087a0. [DOI] [PubMed] [Google Scholar]
  23. TEMPERLI A., TUERLER H., RUEST P., DANON A., CHARGAFF E. STUDIES OF THE NUCLEOTIDE ARRANGEMENT IN DEOXYRIBONUCLEIC ACIDS. IX. SELECTIVE DEGRADATION OF PYRIMIDINE DEOXYRIBONUCLEOTIDES. Biochim Biophys Acta. 1964 Nov 15;91:462–476. doi: 10.1016/0926-6550(64)90076-3. [DOI] [PubMed] [Google Scholar]
  24. VERWOERD D. W., ZILLIG W. A specific partial hydrolysis procedure for soluble RNA. Biochim Biophys Acta. 1963 Mar 26;68:484–486. doi: 10.1016/0006-3002(63)90171-9. [DOI] [PubMed] [Google Scholar]
  25. Vincze A., Henderson R. E., McDonald J. J., Leonard N. J. Reaction of diethyl pyrocarbonate with nucleic acid components. Bases and nucleosides derived from guanine, cytosine, and uracil. J Am Chem Soc. 1973 Apr 18;95(8):2677–2682. doi: 10.1021/ja00789a045. [DOI] [PubMed] [Google Scholar]
  26. WHITFELD P. R., MARKHAM R. Natural configuration of the purine nucleotides in ribonucleic acids; chemical hydrolysis of the dinucleoside phosphates. Nature. 1953 Jun 27;171(4365):1151–1152. doi: 10.1038/1711151a0. [DOI] [PubMed] [Google Scholar]
  27. Wintermeyer W., Zachau H. G. Tertiary structure interactions of 7-methylguanosine in yeast tRNA Phe as studied by borohydride reduction. FEBS Lett. 1975 Oct 15;58(1):306–309. doi: 10.1016/0014-5793(75)80285-7. [DOI] [PubMed] [Google Scholar]
  28. Zimmern D., Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. doi: 10.1073/pnas.75.9.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]

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