Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

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• 作者：Hajime Fukano ; Yosuke Suzuki
• 关键词：Short hairpin RNA ; Fragmentation ; Deoxyribonuclease I ; Klenow fragment ; T4 DNA polymerase ; Single-strand-specific exonuclease
• 刊名：Analytical Biochemistry
• 出版年：2009
• 出版时间：1 February 2009
• 年：2009
• 卷：385
• 期：1
• 页码：80-84
• 全文大小：185 K

文摘

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn²⁺ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg²⁺ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.