通过可控的突出端连接长双链RNA分子。
Joining of long double-stranded RNA molecules through controlled overhangs.
作者信息
Dekker N H, Abels J A, Veenhuizen P T M, Bruinink M M, Dekker C
机构信息
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
出版信息
Nucleic Acids Res. 2004 Oct 8;32(18):e140. doi: 10.1093/nar/gnh138.
Abstract
We describe two methods for creating long (>1 kb) dsRNA molecules with specific, user-controlled overhangs for efficient hybridization and ligation. The two methods create double-stranded RNA (dsRNA) molecules with 5' overhangs or with 3' overhangs using T7 RNA polymerase (T7 RNAP) in transcription reactions of carefully designed PCR products. Primers utilized in the PCR reactions provide the template for the desired dsRNA overhangs. These methods provide complete control of the length and the sequence of the overhangs. This supplies a tool which is particularly lacking in dsRNA biochemistry given the absence of restriction endonucleases active on these substrates.
摘要
我们描述了两种用于创建长（>1 kb）双链RNA（dsRNA）分子的方法，这些分子具有特定的、用户可控制的突出端，以实现高效杂交和连接。这两种方法在精心设计的PCR产物的转录反应中，使用T7 RNA聚合酶（T7 RNAP）创建具有5'突出端或3'突出端的双链RNA（dsRNA）分子。PCR反应中使用的引物为所需的dsRNA突出端提供模板。这些方法可完全控制突出端的长度和序列。鉴于缺乏对这些底物有活性的限制性内切酶，这提供了一种dsRNA生物化学中特别缺乏的工具。
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