A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones.
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").
Borén
J
J
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100, USA. jan-boren.gicd@quickmail.ucsf.edu
Lee
I
I
Callow
M J
MJ
Rubin
E M
EM
Innerarity
T L
TL
D001053:Apolipoproteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011938:Rec A Recombinases / Q000378:metabolism; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000235:genetics
D001053:Apolipoproteins; D018076:DNA, Complementary; D004247:DNA; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes