Nucleic Acids in Biotechnology 189
to be screened separately, which is time consuming. Now more directed methods are standard, where the
DNA is first cloned for ease of manipulation and then deletions, insertions or replacements made.
5.6.1.1 Deletions. Deletions can be created at restriction sites (Section 5.3.1) by cleavage with the
corresponding enzyme and then by treatment for a short period with an exonuclease enzyme. For example,
the exonuclease Bal31 is used to remove both double- and single-stranded DNA from both ends.
Alternatively, the enzyme ExoIII is used to generate single-stranded ends followed by treatment with SI
nuclease to trim the created single strands (Table 5.3). Re-ligation of the two new double-stranded ends
generates deletion mutations of the parent DNA. This method has the serious limitation that deletions can
only be made around restriction sites.
A more general deletion method involves use of synthetic oligonucleotides (Figure 5.16). In this procedure,
an oligonucleotide complementary to the desired site of deletion on the DNA, but not containing the nucleotides
required for deletion, is used as a primer for synthesis of a second DNA strand. In the process of cloning,
mutant DNA segregates from wild type DNA and clones containing mutant the deletion can be selected.
One problem associated with this technique is that bacteria will often attempt to repair the mutagenised
strand because the in vivo-generated DNA strand is methylated. This can result in low yields of the
mutated sequence. Eckstein has developed a reliable method that involves incorporation of phosphorothioate-
modified nucleotides (Section 4.4.3) into the in vitro-generated strand. Such nucleotides are more resist-
ant to nuclease degradation, with the result that the unmutagenised DNA strand can be removed by
exonuclease digestion and the gap filled to generate the mutation in both strands (Figure 5.17). Deletion
mutants can also be generated by this method by use of PCR (Section 5.2.2).
Deletion mutants can also be generated by PCR (Figure 5.18). This method relies upon the fact that PCR
primers are tolerant of primer-template mismatch to create a mutation at the priming site in an analogous
way to that shown in Figure 5.17. Unfortunately, this raises the problem that PCR-based mutagenesis can
only make a mutated site at an end of the PCR fragment. However, this problem can be solved by gener-
ating two PCR products sharing a common central mutated region (Figure 5.18). Denaturation and anneal-
ing of these two products, followed by extension of the duplex with TaqDNA polymerase yields a larger
product with the mutated site in the centre.
5.6.1.2 Insertions. Insertions may be generated by ligation of a synthetic oligonucleotide duplex into
a restriction site after cutting with the appropriate restriction enzyme. Sequence additions at other sites can
Figure 5.16 Oligonucleotide site-directed deletion mutagenesis