An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.

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 insertio...

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Autores principales:	Glen John McIntyre, Jennifer Lynne Groneman, Anna Tran, Tanya Lynn Applegate
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2008
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5652a296c014455780fc3136aac76230
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spelling	oai:doaj.org-article:5652a296c014455780fc3136aac762302021-11-25T06:18:22ZAn infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.1932-620310.1371/journal.pone.0003827https://doaj.org/article/5652a296c014455780fc3136aac762302008-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19043584/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Vector 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.Glen John McIntyreJennifer Lynne GronemanAnna TranTanya Lynn ApplegatePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 3, Iss 11, p e3827 (2008)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Glen John McIntyre Jennifer Lynne Groneman Anna Tran Tanya Lynn Applegate An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
description	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.
format	article
author	Glen John McIntyre Jennifer Lynne Groneman Anna Tran Tanya Lynn Applegate
author_facet	Glen John McIntyre Jennifer Lynne Groneman Anna Tran Tanya Lynn Applegate
author_sort	Glen John McIntyre
title	An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
title_short	An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
title_full	An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
title_fullStr	An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
title_full_unstemmed	An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.
title_sort	infinitely expandable cloning strategy plus repeat-proof pcr for working with multiple shrna.
publisher	Public Library of Science (PLoS)
publishDate	2008
url	https://doaj.org/article/5652a296c014455780fc3136aac76230
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An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.

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