Site-Directed Mutagenesis

About[]

Site-directed mutagenesis is one of the most important laboratory procedures used in the mutation of DNA sequences. It is a method used to make intentional changes to double stranded plasma DNA of genes and genes products, particularly in order to study the structure and activity of DNA, RNA, and protein molecules (1). More specifically, it has been used to understand signaling pathways, drug resistance mechanisms, and promoter DNA binding site identification (2). It is also useful in mutation screening and selecting as well as introduction and removal of restriction endonuclease sites or tags (3).

Origins & Development of Method

Kunkel1 — 1. sDNA plasmid is transformed into ung-bacteria, converting all T's to U's. Ex: GC base pair is targeted for mutagenesis. The U-containing plasmid is converted to single-stranded DNA. Souce: Davidson College Biology Department (4).

The original method of site-directed mutagenesis, developed by Michael Smith (9), involved a technique that reduced the need to select for mutants. It is obtained by using a DNA template containing uracil in place of thymine.

Kunkel2 — 2. Target plasmid is used as a template to produce a second strand of DNA containing the desired mutation and only T's. Source: Davidson College Biology Department.

This template allows for normal coding for in vitro reactions typical of site-directed mutagenesis protocols, but is not fully active until it is transferred into a wild-type E. coli host cell (5). More specifically, the fragment of DNA desired for mutation is first inserted into a phagemid, a plasmid containing an f1 origin of replication that is used as a vector. The plasmid is then transformed into a strain of E. coli that is deficient in two separate enzymes, dUTPase (dut) and uracil deglycosidase (ung). Both of these enzymes have roles in DNA repair, protecting bacterial chromosomes from mutations due to spontaneous removal of an amine group from dCTP, converting it to

Kunkel3 — 3. Hybrid plasmid is transformed into wild-type bacteria that convert the original U-containing strand of DNA into a strand of T-containing DNA that is complement of the mutated sequence (in green). The original GC base pair has been converted to a new TA base pair. Source: Davidson College Biology Department (4).

dUTP. Without dUTPase, there is no breakdown of dUTP, resulting in high cellular dUTP levels. Without uracil deglycosidase, there is no removal of uracil from newly synthesized DNA. As a result, the mutant E. Coli may misincorporate dUTP instead of dTTP while replicating the phage DNA. This results in single-stranded DNA containing some uracils, called ssUDNA. The ssUDNA is then extracted from the bacteriophage and then used for mutagenesis. An oligonucleotide that contains the desired mutation is used to extend the primers, forming DNA consisting of one non-mutated strand with dUTP and one mutated strand containing dTTP. This DNA is then transformed into a wild-type strain of E. Coli (possessing dut and ung genes), allowing the DNA strain containing uracil to degrade, resulting in DNA only consisting of the mutated strand (1).

Today, there are various methods of site-directed mutagenesis as a result of technological advances and scientific breakthroughs throughout time.

Cassette mutagenesis: This method does not involve primer extension through DNA polymerase, but instead synthesis and insertion of a DNA fragment into a plasmid. A restriction enzyme is then added to cleave a site within the plasmid and allow for ligation of the complementary oligonucleotides containing the mutation. This method can generate mutants at nearly 100% efficiency, but is limited by the number of available restriction sites at each end of the desired sites (1).

PCR site-directed mutagenesis: The limitation of available restriction sites by cassette mutagenesis may be relieved with the polymerase chain reaction using oligonucleotide primers, meaning that a larger fragment may then be generated, covering two restriction sites. The amplification in PCR produces a fragment containing the desired mutation in enough quantity that it may be separated from the original, unmated plasmid through gel electrophoresis. The plasmid can then be inserted in its original state (1).

Whole plasmid mutagenesis: Whole plasmid mutagenesis: Other highly efficient site-directed mutagenesis techniques have been simplified and made commercially available. One example is the Quikchange method, using a pair of complementary mutagenic primers to amplify an entire plasmid in a thermocycling reaction by using certain DNA polymerases. This generates a nicked, circular DNA. The template DNA is then eliminated through digestion by a restriction enzyme that is specific for methylated DNA. This results in digestion of the template plasmid while the mutated plasmid is left untouched (1).

Protein Engineering[]

Tide pods SM 223x287 — Tide is one of many companies that uses subtilisin in their detergents. Source: www.tide.com.

Site-directed mutagenesis can be used to alter the activities of proteins in order “improve” them for various practical uses. This is otherwise known as protein engineering. Desired improvements may include increased thermostability, reduced negative feedback inhibition, altered substrate and pH ranges, and more (8). Protein engineering has many commercial applications in which it allows for production of mutant forms tailored for many different possibilities. One example is common laundry detergents containing subtilisin, a protein-digesting enzyme whose wild-type form contains a methionine which can be oxidized by bleach, highly decreasing protein activity in the process (6). This renders the detergent useless when used with bleach. This methionine may be replaced by residues such as alanine, making it resistant to oxidation and keeping the protein active while in the presence of bleach (7).