Chapter 9 Applications of Recombinant DNA Technology

1. Mutagenesis by mutagens often results in multiple mutations, complicating analysis, and the mutation affecting a particular gene is random, rather than directed. Site-specific mutagenesis is a more directed approach.
2. Many procedures have been developed for site-specific mutagenesis, often using PCR (polymerase chain reaction). An example is shown in Figure 9.1.
a. This method uses four primers:
i. Primers are needed for each end of the sequence to be amplified, as in normal PCR (primers 1 and 2 in the figure).
ii. A pair of complementary primers, 1M and 2M, that create the mutation by matching the target sequence except for where the mutation is desired.
iii. Two PCR reactions are done, one with primers 1 and 1M, the other with primers 2 and 2M.
iv. Primers are removed, the two PCR reactions are mixed, and the DNA denatured and allowed to reanneal.
v. Some reannealing will result in strands capable of producing the full-length product in a PCR reaction.
vi. Amplification using primers 1 and 2 produces mutant full-length molecules that can be transformed into a cell to replace the wild-type sequence.