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Nucleic Acids Research, 1977, Vol. 4, No. 10 3563-3572
© 1977

Articles

A two-dimensional thin layer chromatographic procedure for the sequential analysis of oligonucleotides employing tritium post-labeling

Ellson Y. Chen and Bruce A. Roe

Department of Chemistry, Kent State University Kent, OH 44242, USA

Received July 21, 1977. Two dimensional PEI-cellulose thin layer chromatography can resolve sequentially degraded oligonucleotide fragments of tRNA. This technique entails the sequential degradation of the oligo-nucleotide with snake venom phosphodiesterase in the presence of bacterial alkaline phosphatase, and periodate oxidation followed by tritiated sodium borohydride reduction of the 3' terminal nucleoside. Subsequently the tritiated oligonucleotide fragments were resolved by two dimensional PEI-cellulose TLC. The results of these experiments indicate that, in some cases, the complete nucleotide sequence of a large oligonucleotide fragment may be determined by interpretation of the observed mobility shifts, thereby eliminating the need for additional analysis of the oligo-nucleotide. In addition, the use of two-dimensional rather than one-dimensional resolution of the tritium labeled fragments allows for a complete separation of any interfering background spots from the sequentially degraded oligonucleotides.

This procedure was applied to the complete nucleotide sequence analysis of several ribonuclease T1 and ribonuclease A digestion products from human placenta tRNA val.


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