For our purposes, we will define site-directed mutagenesis as any technique for introducing specific base-pair changes at specific locations within a target DNA sequence such as a gene. In general, changes are limited in extent - most often just one or two base pairs - with the intention of changing, for example, a restriction site, a codon, or the sequence of some regulatory element. Occasionally, more extensive changes can be made such as, for example, introducing six histidine codons so that a His-tagged protein my be expressed and isolated.
This approach to mutagenesis was pioneered by the late Michael Smith at the Univerity of British Columbia. He shared the 1993 Nobel Prize in Chemistry for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. Kary Mullis was the co-recipient. He received the prize for for his invention of the polymerase chain reaction (PCR) method
The general strategy for introducing site-specific mutations is shown in the following figure (from the Promega website):
In this strategy, a piece of DNA is first cloned in a suitable vector. A ssDNA copy of the recombinant plasmid is then prepared to which is annealed an oligonucleotide containing the desired sequenced changes. (Sometimes additional oligonucleotides are used as suggested in this figure.) The annealed DNA now serves as a template for DNA polymerase and the resulting heteroduplex DNA is then transformed into E. coli where the mutated clones will be identified.
In theory, if all goes well then 50% of the clones will contain the original sequence and 50% will contain the mutated sequence. In practice, the bias lies greatly in favour of the original sequence. This is due to inefficiencies in synthesising the complementary DNA strand, primer annealing improperly to other sequences in template DNA, and to the host cell repair systems which recognise the heteroduplex and remove the mutation that you are attempting to introduce.
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A number of techniques have been devised to help improve the efficiency site-directed mutagenesis.
Incorporation of deoxyuridine
In this technique, the cloning vector is prepared in a strain of E. coli carrying mutations in two genes: dut and ung. dut enodes dUTPase which normally converts dUTP to dUMP and pyrophophate. This activity prevents the misincorporation of uracil into DNA. ung encodes uracil N-glycosylase whose activity removes uracil from DNA. Recall that uracil can be generated as a result of oxidative deamination of 5-methyl cytosine so it's presence in DNA is generally mutagenic and is normally repaired. By using a template containing uracil for site-directed mutagenesis, the template is theoretically marked for repair. Hence, after oligonculeotide annealing synthesis of the complementary DNA and transformation into a dut+ ung+ strain of E. coli, the original strand will be repaired and the complementary strand will be replicated.
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Elimination of a unique restriction site
In this technique, the cloning vector carries a unique restriction site that can be removed by site-directed mutagenesis. Once again, two primers are used - one to eliminate the restriction site and one to mutate the target of choice. After transformation of the heteroduplex DNA, a small culture is grown from which plasmid DNA is prepared. This is then digested with the restriction enzyme whose site has been mutated. Plasmids that still contain the original restricion site will be linearised. These plasmids represent failed attempts at mutagenesis. Plasmids that no longer contain the original restriction site will not be linearised. However, since circular DNA transforms E. coli with a much higher efficiency than linear DNA, this step selects for circular plasmids that will transform E. coli and which can be identified after a subsequent transformation.
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Repair-deficient host strains
E. coli strains that are mutS- are often used to improve the efficiency of mutagenesis. These strains cannot discriminate between the two DNA strands of a heteroduplex. The use of a mutS- strain helps to ensure that the efficiency of mutagenesis should reach the theoretical 50%.
Positive antibiotic selection
In this technique, the cloning vector carries a defective antibiotic resistance gene that can be repaired by site-directed mutagenesis. Thus, two primers are used - one for the antibiotic resistance gene and one to mutate the target of choice. If the antibiotic resistance gene primer anneals properly, the resulting cells will now be resistant. If that happens, then it is likely that the target mutagenic primer should also have annealed correctly.
Once commercial site-directed mutagenesis system carries this further by mutating one antibiotic resistance gene while simultaneously repairing another. This allows for sequential site-directed mutagenesis to be performed.
Here are some other web sites containing information on site-directed mutagenesis:
| Site-Directed Mutagenesis | By Neal Cosby and Scott Lesley Promega Corporation |
| Site-directed mutagenesis | In Chapter 9: Biotechnology and Proteomics of the Molecular Biology Web Book at Web-Books.com |
| Transformer™ Site-Directed Mutagenesis System |  This Clontech system uses both a unique restriction
enzyme system and a repair-deficient host strain. |
| Oligonucleotide-directed mutagenesis | Dr. Stanley Maloy's Mcbio 316 web notes (University of Illinois at Urbana-Champaign). |
