Use of Quantitative Ligation-Mediated Polymerase Chain Reaction to Detect Gene Targeting by Alkylating Oligodeoxynucleotides

The authors describe the preparation and use of a unique class of reactive oligodeoxynucleotides (ODNs) that can sequence, specifically bind to, and alkylate complex genomic DNA under physiological conditions. The alkylating event, which is detected using a quantitative version of the ligation-mediated polymerase chain reaction (LM-PCR) (1 ,2 ), acts as a marker for complex formation, and permits an estimation of the gene targeting frequency. Because alkylation of the DNA can take place within either a classical triple-stranded complex (3 ) or a recombinase-stabilized synaptic joint (4 ), two different strategies for recognizing DNA can be directly compared. The ability to detect and quantify an early, perhaps rate limiting step, in genetic targeting should be useful in efforts to better understand and improve this important technique.