Batchwise Purification of Specific tRNAs by a Solid-Phase DNA Probe

✍ Scribed by H. Tsurui; Y. Kumazawa; R. Sanokawa; Y. Watanabe; T. Kuroda; A. Wada; K. Watanabe; T. Shirai

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 608 KB
• Volume: 221
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1393

✦ Synopsis

A simple and efficient method for purifying a specific tRNA in a single microcentrifuge tube was developed. Oligodeoxyribonucleotides (about 30 mer) with sequences complementary to the 3' side of target tRNAs were synthesized with an aminohexyl linker at the 5' end, immobilized on a silica gel at a high concentration, and used as a solid-phase probe. A mixture of tRNAs was added to a suspension of the solid-phase probe in 2.4 M tetraethylammonium chloride and incubated for 10-30 min. Only a target tRNA hybridized with the immobilized probe at appropriate temperatures and was eluted out by heating. The solid-phase probe showed a large hybridization capacity (up to 17 A260 units/g dry gel) and specific and quantitative recovery of the target tRNA. The intactness of recovered tRNAs was ascertained by both Donis-Keller sequencing and aminoacylation experiments. These features show the usefulness of the solid-phase probe method as a reliable tool for purifying tRNAs whose gene sequences are known.