Oligonucleotide-directed mutagenesis involves the incorporation of a mutant oligonucleotide into one strand of plasmid DNA. After DNA replication of this heteroduplex plasmid, any progeny plasmids that arise by replication of the wild-type strand will be homozygous for the wild-type allele, and any plasmids that arise by replication of the mutant strand will be homozygous for the mutant allele. The resulting population of plasmids contains both wild-type and mutant plasmids that may only differ by a single base-pair mutation in a sequence which lacks an easily assayed phenotype. Furthermore, when introduced into cells the mismatch repair system often repairs the mutated base to the complementary base in the wild-type strand before it has a chance to replicate, so the mutant plasmids are underrepresented relative to the wild-type plasmids. The trick to oligonucleotide-directed mutagenesis is to identify the desired mutants.
There are many different methods of oligonucleotide-directed mutagenesis, but most are minor variations on two ways of enriching for the desired mutant plasmids: (1) methods which destroy the wild-type template strand of DNA and thus favor replication of the mutant strand, and (2) methods which simply allow the wild-type and mutant strands of DNA an equal chance of replicating. The dut ung method is an example of the first approach, and the mutS method is an example of the second approach.
The DNA template is obtained from plasmids or phage grown in a dut ung mutant of E. coli. The dut gene encodes dUTPase which normally degrades dUTP. An elevated concentration of dUTP accumulates in dut strains, resulting in incorporation of U in place T at some positions during DNA replication. The ung gene encodes uracil N-glycosylase which normally removes U from DNA. Thus, in the double mutant U is occasionally incorporated into DNA and this error is not repaired. Because U has the same base pairing properties and the same coding properties as T, incorporation of U into DNA in place of T is not mutagenic.
A mutagenic oligonucleotide is annealed to a ssDNA template obtained from the dut ung strain. This oligonucleotide serves as a primer for in vitro DNA replication. The in vitro DNA replication reaction contains DNA polymerase, dATP, dTTP, dGTP, and dCTP but no dUTP, so no U is incorporated into the newly synthesized strand of DNA. After synthesis of the second strand of DNA is completed, the ends are covalently joined by DNA ligase. The resulting dsDNA consists of the template strand which contains U residues, and the newly synthesized strand which contains the mutant bases present in the oligonucleotide primer and does not contain any U residues.
This dsDNA is then transformed into an ung+ recipient cell. The uracil N-glycosylase recognizes the U residues in the DNA, and excises the U leaving apyrimidinic (AP) sites in the template strand. Presence of AP sites makes the DNA strand biologically inactive because it cannot be replicated by DNA polymerase and the DNA is cut at the AP sites by specific endonucleases. Hence, when the dsDNA is introduced into the ung+ recipient , only the mutant strand will be replicated.
Note that the dut ung method provides an enrichment for the mutant plasmids, not a selection -- typically only about 50% of the resulting plasmids carry the mutation introduced on the oligonucleotide.
The mutS gene encodes an essential componant of the methyl-directed mismatch repair system. Thus, a mutS mutation in the recipient prevents repair of the mutant base to the wild-type sequence, so that after DNA replication about 50% of the plasmids will be mutant and 50% will be wild-type.
An very clever variation of the mutS approach was developed by scientists at Promega and is marketed as the "Altered Sites Kit". A figure of this approach is shown below and a more detailed description of this technique can be found on the Promega WWW site.
Questions to ponder:
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Last modified July 23, 2002