PMID: 8938437Nov 1, 1996Paper

A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones

Genome Research
J BorénT L Innerarity

Abstract

This study addresses two important technical problems: how to perform targeted alterations such as site-directed mutagenesis and deletions in large fragments of DNA and how to construct full-length genes from two partly overlapping bacterial artificial chromosome (BAC) plasmids. Given the size and the lack of convenient unique restriction sites in these large-insert bacterial clones, these are nontrivial tasks. Here we describe a simple and efficient protocol based on RecA-assisted restriction endonuclease (RARE) cleavage, a method that enables sequence-specific cleavage of genomic DNA. The same protocol has been used with minor modifications to introduce site-specific mutations into an apolipoprotein-B 90-kb P1 clone, to generate deletions in a 160-kb BAC, and to generate a 160-kb BAC containing the complete 92-kb gene for low-density lipoprotein-related protein-1 (LRP-1) from two smaller overlapping BACs ("BAC marriage").

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Citations

Jun 5, 2003·Genomics·Elena G BochukovaAnthony P Monaco
Feb 1, 1997·Current Opinion in Biotechnology·W Szybalski
Oct 24, 2003·Molecular and Cellular Neurosciences·Kathrin DethleffsenMichael Meyer
Apr 16, 1998·Proceedings of the National Academy of Sciences of the United States of America·L J Ferrin, R D Camerini-Otero
Feb 5, 2011·Atherosclerosis·Ana Paula Queiroz MelloNágila Raquel Teixeira Damasceno
Aug 2, 2001·Nature Biotechnology·F StoriciM A Resnick
Oct 1, 2011·Circulation Research·Malin C LevinJan Borén
Dec 25, 2000·The Journal of Biological Chemistry·J BorénT L Innerarity
Jun 19, 2002·The Journal of Biological Chemistry·Christofer FloodJan Borén
May 8, 1998·Methods : a Companion to Methods in Enzymology·G E Herman

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