sites that make them incompatible with some vectors. The development of the polymerase
chain reaction (PCR) (Fig. 7.10) in 1985, by Kary Mullis, revolutionized the manipulation
of DNA, facilitating the inclusion of restriction sites in positions flanking a gene, or its pro-
moter, facilitating cloning as well as the removal of internal restriction sites, while maintain-
ing the integrity of the gene. PCR amplifies specific DNA sequences in a test tube and also
allows the sequences to be changed. Despite these improvements, the production of con-
structs is laborious, and inappropriately positioned restriction sites are still a major factor
that hinders vector construction.
7.3.1 Site-Specific DNA Recombination
Several strategies have been developed to overcome the difficulties associated with conven-
tional cloning. These have been compounded by the demands of the numerous functional
Figure 7.10.Polymerase chain reaction is a technique that allows a chosen region of DNA to be
amplified in vitro by separating the double-stranded DNA template into two strands by denaturation
and incubating with oligonucleotide primers and DNA polymerase to synthesize a complementary
strand of each. The primers can be designed to incorporate restriction enzyme recognition sites or
any other recognition sequence to facilitate the cloning of PCR fragments. Repeated cycles of dena-
turation, primer annealing, and extension (DNA synthesis with DNA polymerase) allow the targeted
region of DNA to be amplified many thousands of times. This tool is frequently used in biotechno-
logy, forensics, medicine, and genetic research to amplify DNA fragments.
7.3. GREATER DEMANDS LEAD TO INNOVATION 171