Site-directed mutagenesis
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Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. In general, site-directed mutagenesis requires that the wild type gene sequence be known.
This technique is also known as site-specific mutagenesis or oligonucleotide-directed mutagenesis.
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[edit] Invention
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Site-directed mutagenesis using oligonucleotides was first described in 1978[1]. Michael Smith, its pioneer, shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis who developed the PCR technique.
In 1987 Kunkel et al. introduced an improvement to the technique that eliminated the need for phenotypic selection. The plasmid to be mutated would be transformed into an E. coli deficient in two genes, dUTPase and uracil deglycosidase. The former would prevent the breakdown of dUTP, a nucleotide that replaces dTTP in RNA, resulting in an abundance of the molecule, and deficiency in the latter would prevent the removal of dUTP from newly synthesized DNA. As the double-mutant E. coli replicates the up-taken plasmid, its enzymatic machinery incorporates the dUTP, resulting in a distinguishable copy. This copy is then extracted and incubated with an oligonucleotide containing the desired mutation, which attaches by base pair hydrogen bonding to the complementary wild type gene sequence, as well as the Klenow enzyme, dNTPs, and DNA ligase. The reaction essentially replicates the dUTP-containing plasmid using as primer the oligonucleotide, giving a nearly identical copy. The essential differences being that the copy contains dTTP rather than dUTP, as well as the desired mutation. When the chimeric double-stranded plasmid, containing the dUTP, unmutated strand and the dTTP, mutated strand, is inserted into a normal, wild-type E. coli, the dUTP-containing strand is broken down, whereas the mutation-containing strand is replicated.
[edit] Basic mechanism
The basic procedure starts with synthesizing a short DNA primer, containing the desired base change. Next this synthetic primer has to hybridize with a single-stranded DNA containing the gene of interest. Third, the single stranded fragment is extended using DNA polymerase, which copies the rest of the gene. Fourth, the obtained double stranded molecule is introduced into a host cell and cloned. Fifth, mutants are selected for.
- see also non-public domain figure
[edit] Cassette Mutagenesis
Cassette mutagenesis involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of an oligonucleotide containing the mutation in the gene of interest to the plasmid. Usually the restriction enzyme that cuts at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another.
[edit] PCR site-directed mutagenesis
The same result can be accomplished using PCR with oligonucleotide "primers" that contain the desired mutation. As the primers are the ends of newly-synthesized strands, by engineering a mis-match during the first cycle in binding the template DNA strand, a mutation can be introduced. Because PCR employs exponential growth, after a sufficient number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques.
For plasmid manipulations, this technique has largely been supplanted by a PCR-like technique where a pair of complementary mutagenic primers is used to amplify the entire plasmid. This generates a nicked, circular DNA which can undergo repair by endogenous bacterial machinery. However, this process does not amplify the DNA exponentially, rather, linearly. Yields are complicated by the fact that the product DNA must undergo the nick repair and is not supercoiled, resulting in lowered efficiency of transformation in bacteria. Finally, the product DNA is of the same size as the plasmid. Therefore, the template DNA must be eliminated by enzymatic digestion with a restriction enzyme specific for methylated DNA. The template, which for this technique should be biosynthesized will be digested, but the mutated plasmid is preserved because it was generated in vitro and is therefore unmethylated.
[edit] See also
Oligonucleotide- based mutagenesis is the most commonly used method to introduce mutations in coding sequence.
Deletion mutagenesis is a simple technique in which DNA sequences are removed from either end of the cDNA clone using an enzyme such as exonucleasesIII(digest nucleotides progressively from a RECESSED DNA strand in the 3'-t 5' direction).
[edit] In vivo site directed mutagenesis methods
- Delitto perfetto[2]
- Transplacement "pop-in pop-out"
- Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker
- Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions
- In vivo site-directed mutagenesis with synthetic oligonucleotides[3]
[edit] References
- ^ Hutschison, C.A., Philipps, S., Edgell, M.H., Gillham, S., Jahnke, P., Smith, M. (1978) Mutagenesis at a Specific Position in a DNA Sequence. J. Biol. Chem. 253: (18) 6551-6560
- ^ Storici F., Resnick MA. (2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol. 409:329-45.
- ^ Storici F., Resnick MA. (2003) Delitto perfetto targeted mutagenesis in yeast with oligonucleotides. Genet Eng. 25:189-207
