Bio470: Site-Directed Mutagenesis

Site-directed mutagenesis

The principle of site-directed mutagenesis is that a mismatched oligonucleotide is extended, incorporating the "mutation" into a strand of DNA that can be cloned. I will present a number of current methods in use (including some course content downloaded from other commercial or university sites).

The 4 oligo method
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. Or, we can continue to manipulate the DNA by PCR.

Site directed mutagenesis (inside out)
This is how we're handling the mutagenesis problem in our laboratory:

We start with a circular plasmid, and use two oligonucleotides to change a small region by PCR (see asterisk). The 5' ends of the oligonucleotides are shown not annealed - they do not base pair because they are mutagenized. The two oligos are situated in such a way that they re-copy the entire plasmid.

That is like deciding to go to Los Angeles for the day, but instead of heading down the 405 you go up the Pacific Coast Highway to Alaska, snowshoe over to Denmark, hop a train to Capetown, boat over to Tierra del Fuego, and bicycle up through South and Central America to Los Angeles! It's the long way around, but in this example it makes sense because it means you don't need to combine two pieces for cloning. What you obtain in the end is a linear fragment, suitable for reclosure and cloning:

Resources:
Protein engineering and cleaner clothes (ASU)

Oligonucleotide-directed Mutagenesis (Maloy)

The Altered States Method (Promega)

In-class problems

The 4 oligo method	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. Or, we can continue to manipulate the DNA by PCR.
Site directed mutagenesis (inside out)	This is how we're handling the mutagenesis problem in our laboratory: We start with a circular plasmid, and use two oligonucleotides to change a small region by PCR (see asterisk). The 5' ends of the oligonucleotides are shown not annealed - they do not base pair because they are mutagenized. The two oligos are situated in such a way that they re-copy the entire plasmid. That is like deciding to go to Los Angeles for the day, but instead of heading down the 405 you go up the Pacific Coast Highway to Alaska, snowshoe over to Denmark, hop a train to Capetown, boat over to Tierra del Fuego, and bicycle up through South and Central America to Los Angeles! It's the long way around, but in this example it makes sense because it means you don't need to combine two pieces for cloning. What you obtain in the end is a linear fragment, suitable for reclosure and cloning:
Resources:	Protein engineering and cleaner clothes (ASU) Oligonucleotide-directed Mutagenesis (Maloy) The Altered States Method (Promega)