Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation
Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation
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DOI：
10.1093/jb/mvu089
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发表时间：
2015-05-01
影响因子：
2.7
通讯作者：
Hori, Hiroyuki
中科院分区：
文献类型：
--
作者：
Kazayama, Ai;Yamagami, Ryota;Hori, Hiroyuki
固相DNA探针法通常用于tRNA的纯化，该方法将目标转移RNA （tRNA）与互补的DNA寡聚物杂交。然而，用这种方法从嗜热菌中纯化trna并不容易，因为它们的熔融温度很高。为了克服这一问题，先前有报道使用四烷基铵盐[Yokogawa， T., Kitamura， Y., Nakamura， D., Ohno， S., and Nishikawa， K.（2010）优化了在四烷基铵盐存在下纯化耐热trna的基于杂交的方法。中国生物医学工程学报[j]。在这项研究中，我们初步设计了一个使用四烷基铵盐的大规模净化系统。与之前的方法相比，tRNA的产率提高了10倍以上，减少了人工步骤。然而，由于生物素化DNA探针的脱落，柱的恶化非常迅速。因此，我们设计了一种共价DNA固定方法，将5'-氨基己基（dT）(8)低聚物固定在n -羟基琥珀酰亚胺活化的琼脂糖上，然后将含有tRNA和重复a(8)序列的DNA低聚物退火。用Klenow酶在柱上合成tRNA纯化探针序列。这种DNA固定方法使我们能够重复使用色谱柱，并用加热的缓冲液清洗色谱柱。因此，与以前使用生物素化DNA探针的方法相比，这种DNA固定方法是经济的。
The solid-phase DNA probe method, in which a target transfer RNA (tRNA) is hybridized with a complementary DNA oligomer, is generally used for tRNA purification. However, purification of tRNAs from thermophiles by this method is not easy because of their high melting temperatures. To overcome this problem, the use of tetraalkylammonium salts was previously reported [Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89]. In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)(8) oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A(8) sequences was annealed. The probe sequence for tRNA purification was synthesized in column with Klenow enzyme. This DNA fixation method enabled us to use the column repeatedly and to wash the column with warmed buffers. Thus, this DNA fixation method is economical as compared with the previous method using the biotinylated DNA probe.