Site directed mutagenesis

It is often the case in genetic engineering that one wishes to make very precise changes in the sequence of a DNA molecule. Here's an example:

Creating a mutated allele that can be reintroduced into an organism.
Suppose you want to create a point mutation in a gene, at the position marked with an "*" in the figure:

The ways of doing this in the old days were unspeakable, but now we can simply get on the phone and order four oligonucleotides; two of which are flanking and two of which cover and introduce the mutation into the amplified material:

We perform two PCR reactions to obtain the two halves of our final product, and combine them in a third reaction, using the two "outside" oligonucleotides to generate a chimeric product.

How does this happen? During the PCR process, the right side of the first molecule can prime the synthesis from the left side of the second.

Now we can simply cut the PCR product with EcoRI and BamHI, and drop it into the vector, in place of the original version.

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The Altered States® method.

Here's a way to use selective markers in a plasmid, in order to help you isolate genes with specific mutations. It isn't based on PCR (but you can see that it could be!).

Start with a plasmid carrying a defective selectable marker (e.g. Amp)
Link the mutation you are making elsewhere in the plasmid to a correction of the defective selectable marker.
Select for correction of the selectable marker, and you are likely to also find plasmids with your specific mutation introduced as well
Sound's easy? Here's a diagram that should help, from the Promega site.

Figure 2 (above) outlines the mutagenesis procedure, which can be performed on either single- or double-stranded DNA templates with efficiencies often greater than 90%. Figure 3 (below) shows conversion of lacZ- to lacZ+ using a derivative of pALTER(TM)-1 Vector. For selection, both the lacZ mutagenic oligonucleotide and the Tetracycline Knockout Oligonucleotide were used in addition to the Ampicillin Repair Oligonucleotide. Ampicillin resistant colonies were screened for beta-galactosidase activity and resistance to tetracycline. Greater than 80% of the ampicillin resistant colonies contained both the lacZ and tetracycline mutations. A second round of mutagenesis can be performed on one of these lacZ+ tets mutants using the Ampicillin Knockout Oligonucleotide with the Tetracycline Repair Oligonucleotide for selection. Tetracycline resistant colonies are screened for sensitivity to ampicillin. By the alternate use of the two antibiotic resistances encoded by the vectors, it is possible to generate an indefinite number of mutations without subcloning the insert into another vector. This property is particularly useful for confirming the effect of a mutation by restoring the wild-type sequence through another round of mutagenesis. Mutagenesis of a pALTER(TM)-1 derivative. A lacZ- derivative of pALTER(TM)-1 was converted to lacZ+ using the recommended double-stranded procedure (6). The Ampicillin Repair Oligonucleotide and Tetracycline Knockout Oligonucleotide were used in addition to a lacZ repair oligonucleotide. Ampicillin resistant colonies of JM109 were replica plated onto plates containing either ampicillin, IPTG, and X-gal or only tetracycline. Repair of the lacZ- mutation results in a blue phenotype on plates containing ampicillin, IPTG and X-gal. Inactivation of the tetracycline gene from the use of the Tetracycline Knockout Oligonucleotide results in no growth on the tetracycline plate. Promega Corporation: http://www.promega.com/pnotes/46/2259a/2259a.html