AI Article Synopsis
- Several techniques exist for creating short hairpin RNA (shRNA) expression libraries used in RNA interference for genetic analysis.
- Our method utilizes 25- to 35-bp DNA fragments produced by fragmenting double-stranded DNA (dsDNA).
- We found that using DNase I with Mg(2+) for nicks prior to blunting with the Klenow fragment was more effective for producing these DNA fragments, although it initially led to some unwanted fusion of fragments, which we were able to reduce using single-strand-specific exonucleases.
Article Abstract
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 Mn(2+) followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg(2+) 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.
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| http://dx.doi.org/10.1016/j.ab.2008.10.026 | DOI Listing |
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