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Recombining overlapping BACs into a single larger BAC

BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large...

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Autores principales: Kotzamanis, George, Huxley, Clare
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC317331/
https://www.ncbi.nlm.nih.gov/pubmed/14709179
http://dx.doi.org/10.1186/1472-6750-4-1
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author Kotzamanis, George
Huxley, Clare
author_facet Kotzamanis, George
Huxley, Clare
author_sort Kotzamanis, George
collection PubMed
description BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. RESULTS: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. CONCLUSION: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones.
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spelling pubmed-3173312004-01-23 Recombining overlapping BACs into a single larger BAC Kotzamanis, George Huxley, Clare BMC Biotechnol Methodology Article BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. RESULTS: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. CONCLUSION: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones. BioMed Central 2004-01-06 /pmc/articles/PMC317331/ /pubmed/14709179 http://dx.doi.org/10.1186/1472-6750-4-1 Text en Copyright © 2004 Kotzamanis and Huxley; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
spellingShingle Methodology Article
Kotzamanis, George
Huxley, Clare
Recombining overlapping BACs into a single larger BAC
title Recombining overlapping BACs into a single larger BAC
title_full Recombining overlapping BACs into a single larger BAC
title_fullStr Recombining overlapping BACs into a single larger BAC
title_full_unstemmed Recombining overlapping BACs into a single larger BAC
title_short Recombining overlapping BACs into a single larger BAC
title_sort recombining overlapping bacs into a single larger bac
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC317331/
https://www.ncbi.nlm.nih.gov/pubmed/14709179
http://dx.doi.org/10.1186/1472-6750-4-1
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