NDLI: An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Content Provider	PubMed Central
Author	Mcintyre, Glen John Groneman, Jennifer Lynne Tran, Anna Applegate, Tanya Lynn
Editor	Zhang, Baohong
Copyright Year	2008
Abstract	Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.
Related Links	http://dx.doi.org/10.1371/journal.pone.0003827
Starting Page	3827
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	11
Volume Number	3
Language	English
Publisher	Public Library of Science
Publisher Date	2008-11-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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