Enzymatic production of single-molecule FISH and RNA capture probes
Enzymatic production of single-molecule FISH and RNA capture probes
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DOI：
10.1261/rna.061184.117
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发表时间：
2017-10-01
期刊：
影响因子：
4.5
通讯作者：
Ephrussi, Anne
中科院分区：
文献类型：
--
作者：
Gaspar, Imre;Wippich, Frank;Ephrussi, Anne
与特定靶标杂交的单标记短寡核苷酸阵列彻底改变了RNA生物学，使定量单分子显微镜分析和高效RNA/RNP捕获成为可能。在这里，我们描述了一种简单而有效的方法，允许灵活的功能化廉价的DNA寡核苷酸，通过不同的荧光染料或生物素使用末端脱氧核苷酸转移酶和定制的功能基团共轭双脱氧-UTP。我们表明，（i）寡核苷酸标记的所有步骤-包括缀合、酶促合成和产物纯化-可以在标准生物学实验室中进行，（ii）该过程产生> 90%，通常>95%的标记产物，杂质残留最少，和（iii）寡核苷酸可以用不同的染料或生物素标记，允许单分子FISH、RNA亲和纯化，并进行北方印迹分析。
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.