HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY OF MESSENGER-RNA
HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY OF MESSENGER-RNA
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DOI：
10.1016/0021-9673(91)85017-a
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发表时间：
1991-12-27
期刊：
影响因子：
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通讯作者：
JARRETT, HW
中科院分区：
文献类型：
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作者：
GOSS, TA;BARD, M;JARRETT, HW
使用 N-羟基琥珀酰亚胺化学将 50 聚体胸苷酸 (dT)50 与预装柱内的二氧化硅偶联。所得的 (dT)50-硅胶柱用于解析腺苷酸寡聚物 (dA)19-24，并从酵母菌中分离出 Poly(A) mRNA（信使 RNA）。长度仅相差一个核苷酸碱基的寡聚物很容易被解析。使用 (dT)50- 或 (dT)18- 硅胶，poly(A) mRNA 可在短短 8 分钟内纯化。分离的 Poly(A) mRNA 似乎是全长的，可直接用于 T4 RNA 连接酶以及 RNAse A 和 T1 酶促反应。 (dT)50-硅胶柱用于按尾长度分级总的poly(A) mRNA。虽然分离主要是由于聚 (A) 尾长，但大多数级分似乎包含多个尾长。讨论了这是否代表 RNA 的内在特征或方法的局限性。这些研究表明，千碱基大小范围内的多核苷酸可以在 DNA 硅胶上快速分离并具有良好的分辨率。
A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry. The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces. Oligomers which differed in length by a single nucleotide base were readily resolved. Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min. The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions. The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length. While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths. Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed. These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.