Recombineering with ssDNA

In 2001, Hillary Ellis and colleagues achieved a breakthrough by demonstrating that ssDNA can be used as a recombineering substrate. With a commercial 40-70 base long oligo, precise mutations can be made at a high frequency, typically at 105/108 viable cells, but with some as high as a few percent of the viable cells. Of the three λ Red functions, only Beta is required for recombination with ssDNA. Insight was gained on the mechanism of ssDNA recombination (see below) when it was discovered that of the two complementary oligos that can be used to modify any sequence, the one that corresponds in sequence to the lagging, discontinuously replicated strand is always more efficient, usually by approximately 20-fold.


 © RECOMBINEERING 2016