The PCR method has also found widespread application in forensics for the detection and amplification
of very tiny amounts of DNA, for example at crime scenes, and in DNA mutation detection.
5.3 Enzymes Useful in Gene Manipulation
Gene cloning would not have become possible without the discovery and isolation of a range of enzymes that
act on DNA to enable manipulation of particular sections. One important class of enzymes is the DNA poly-
merases (Section 3.6.1). Other enzymes are involved in cutting the DNA, adding or removing a terminal
phosphate or joining DNA fragments.
5.3.1 Restriction Endonucleases
Bacteria require a system to prevent foreign DNA from being replicated. This is provided by restriction
endonucleases, which recognise and bind to DNA sequences at specific sites and make a double-stranded
cleavage (Section 10.5.1). There are three types of restriction and modification systems, termed I, II and
III in order of their discovery. The names of the enzymes are usually based on the names of the bacteria
from which they are isolated. More than 3600 enzymes have been identified to date (http://rebase.
neb.com/rebase/rebase.html).
174 Chapter 5
Figure 5.5 Steps involved in the polymerase chain reaction (PCR)