Abstract
A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.
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- Amandaraj M. P., Roe B. A. Purification of human placenta phenylalanine, valine, methionine, glucine, and serine transfer ribonucleic acids. Biochemistry. 1975 Nov 18;14(23):5068–5073. doi: 10.1021/bi00694a006. [DOI] [PubMed] [Google Scholar]
- Ando T. A nuclease specific for heat-denatured DNA in isolated from a product of Aspergillus oryzae. Biochim Biophys Acta. 1966 Jan 18;114(1):158–168. doi: 10.1016/0005-2787(66)90263-2. [DOI] [PubMed] [Google Scholar]
- Chang S. H., Kuo S., Hawkins E., Miller N. R. The corrected nucleotide sequence of yeast leucine transfer ribonucleic acid. Biochem Biophys Res Commun. 1973 Apr 16;51(4):951–955. doi: 10.1016/0006-291x(73)90019-3. [DOI] [PubMed] [Google Scholar]
- De Wachter R., Fiers W. Studies on the bacteriophage MS2. IV. The 3'-OH terminal undecanucleotide sequence of the viral RNA chain. J Mol Biol. 1967 Dec 28;30(3):507–527. [PubMed] [Google Scholar]
- Kowalski S., Yamane T., Fresco J. R. Nucleotide sequence of the "denaturable" leucine transfer RNA from yeast. Science. 1971 Apr 23;172(3981):385–387. doi: 10.1126/science.172.3981.385. [DOI] [PubMed] [Google Scholar]
- RajBhandary U. L. Studies on polynucleotides. LXXVII. The labeling of end groups in polynucleotide chains: the selective modification of diol end groups in ribonucleic acids. J Biol Chem. 1968 Feb 10;243(3):556–564. [PubMed] [Google Scholar]
- Randerath E., Randerath K. Ion-exchange thin-layer chromatography. XVI. Techniques for preparation and analysis of oligonucleotides. J Chromatogr. 1967 Dec;31(2):485–499. doi: 10.1016/s0021-9673(01)86099-4. [DOI] [PubMed] [Google Scholar]
- Randerath E., Yu C. T., Randerath K. Base analysis of ribopolynucleotides by chemical tritium labeling: a methodological study with model nucleosides and purified tRNA species. Anal Biochem. 1972 Jul;48(1):172–198. doi: 10.1016/0003-2697(72)90181-9. [DOI] [PubMed] [Google Scholar]
- Randerath K. An evaluation of film detection methods for weak beta-emitters, particularly tritium. Anal Biochem. 1970 Mar;34:188–205. doi: 10.1016/0003-2697(70)90100-4. [DOI] [PubMed] [Google Scholar]
- Randerath K., Chia L. S., Gupta R. C., Randerath E. Structural analysis of nonradioactive RNA by postlabeling: the primary structure of baker's yeast tRNA Leu/CUA. Biochem Biophys Res Commun. 1975 Mar 3;63(1):157–163. doi: 10.1016/s0006-291x(75)80024-6. [DOI] [PubMed] [Google Scholar]
- Randerath K., Randerath E., Chia L. S., Gupta R. C., Sivarajan M. Sequence analysis of nonradioactive RNA fragments by periodate-phosphatase digestion and chemical tritium labeling: characterization of large oligonucleotides and oligonucleotides containing modified nucleosides. Nucleic Acids Res. 1974 Sep;1(9):1121–1141. doi: 10.1093/nar/1.9.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Randerath K., Randerath E. Ion-exchange thin-layer chromatography. XV. Preparation, properties and applications of paper-like PEI-cellulose sheets. J Chromatogr. 1966 Apr;22(1):110–117. doi: 10.1016/s0021-9673(01)97076-1. [DOI] [PubMed] [Google Scholar]
- Roe B. A., Anandaraj M. P., Chia L. S., Randerath E., Gupta R. C., Randerath K. Sequence studies on tRNAPhe from placenta: comparison with known sequences of tRNAPhe from other normal mammalian tissues. Biochem Biophys Res Commun. 1975 Oct 27;66(4):1097–1105. doi: 10.1016/0006-291x(75)90470-2. [DOI] [PubMed] [Google Scholar]
- Sanger F., Brownlee G. G., Barrell B. G. A two-dimensional fractionation procedure for radioactive nucleotides. J Mol Biol. 1965 Sep;13(2):373–398. doi: 10.1016/s0022-2836(65)80104-8. [DOI] [PubMed] [Google Scholar]
- Simsek M., Ziegenmeyer J., Heckman J., Rajbhandary U. L. Absence of the sequence G-T-psi-C-G(A)- in several eukaryotic cytoplasmic initiator transfer RNAs. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1041–1045. doi: 10.1073/pnas.70.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sivarajan M., Gupta R. C., Chia L. L., Randerath E., Randerath K. Tritium sequence analysis of oligoribonucleotides: a combination of post-labeling and thin-layer chromatographic techniques for the analysis of partial snake venom phosphodiesterase digests. Nucleic Acids Res. 1974 Oct;1(10):1329–1341. doi: 10.1093/nar/1.10.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Szeto K. S., Söll D. Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. Nucleic Acids Res. 1974 Jan;1(1):171–181. doi: 10.1093/nar/1.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]