Mutagenesis is the careful alteration of the genetic information of an organism’s genome in such a way that it will result in a mutation. It is the creation of genetic change in the DNA of an organism and, which will result in the production of a population of cells with an entirely new genetic make-up. Generally, mutagenesis is a technique used in molecular biology to create mutant genes, proteins, and organisms. Mutagenesis which can either be spontaneous (natural) or induced (artificial) causes an alteration in the genetic information (or DNA) of an organism. Such a change in the DNA can be passed on from one organism to another and from one generation to another generation. Such mutations or change in an organism’s genome can either be of positive effect or negative effect. In the negative aspect, mutagenesis which can also be a driving force of evolution can result to various genetic diseases and cancer in humans. Such mutations are the causes of a number of genetic diseases (some of which are inheritable) in human beings. Mutagenesis that occurs in the gamete or sex cells (e.g. sperm or ova) of an organism can be passed on from parents to their offspring’s.
But the mutation that occurs in the somatic cells (e.g. skin) of an organism including mutagenesis in cells that have undergone cell differentiation are usually not inherited or passed on to the next generation. Such mutations start and end in the organisms in which it occurred. Mutagenesis can be provoked experimentally in a microbial cell using specific laboratory protocols. Such alterations in the genome of an organism can result to the production of a newly beneficial protein or metabolite with advantageous functions and, which can be transferred to the next generation. Mutagenesis can also be referred to as the defect in the DNA of an organism at the molecular level or the process of achieving transformation in an organism (in this case, a microbial cell). It can occur either in the form of a point mutation, deletion, duplication, translocation or inversion in the base sequence of the organism’s genome.
Mutagens (e.g. radiations and chemicals like bleach) can also stimulate mutagenesis in the cell of an organism. The integrity of a cell’s genome that experienced mutagenesis can actually be repaired through a number of available DNA repair mechanisms (e.g. nucleotide excision repair mechanism-which allows the recognition and removal of damaged DNA and, their subsequent replacement with the correct nucleotide via DNA ligation mechanism). Microbial cells ensures accurate replication of their genome (DNA) during cell division and, keep mutations that spontaneously occurs during such processes to the barest minimum by ensuring adequate repair of DNA anytime mutagenesis occurs. Studies in mutagenesis have helped scientists to better understand the mechanisms and operation of cellular processes of organisms (especially those of the microbial world), and this has helped biologists to undertake research geared towards proper handling of mutagenic risks amongst human population.
Types of mutagenesis
There are basically two primary mutagenesis techniques:
- Site-directed mutagenesis (SDM)
- Random-and-extensive mutagenesis (REM).
Site-directed Mutagenesis (SDM)
SDM is a technique where DNA can be modified at a specific nucleotide location, causing a predetermined amino acid change. These substitution mutations can result in drastic changes in protein conformation and function. This technique requires (1) a DNA template with a target gene, (2) knowledge of the target gene’s nucleotide sequence, and (3) a short primer (commonly 20 to 30 base pairs) complementary to the target sequence that is modified to contain a mismatched nucleotide (typically 1 to 3 base pairs that will cause an amino acid change). The general procedure for SDM is the following:
- Step 1: Separation of the two strands of template DNA. This can be accomplished by heat or an alkali solution.
- Step 2: Addition of the modified DNA primer. Once the DNA primer anneals with a single strand, a DNA polymerase replicates the strand.
- Step 3: The second round of replication yields a mutant DNA strand that can be used to synthesize a modified protein.
Random and Extensive Mutagenesis (REM)
REM is a useful approach when many mutations are desired; however, there is less control over the resulting modifications. This technique has helped map out critical residues of proteins (e.g., EcoRV restriction endonuclease). REM can be accomplished through different methods:
- Primers randomly produced with mismatched bases. In this technique, a set of mutagenic primers with three mismatched bases at a single base position are synthesized in the same reaction. These sets of primers are then used to synthesize mutant DNA with three different mutations at the same base position. Other than this, a primer can be synthesized using an ambiguous base (e.g., deoxyinosine), which is capable of base-pairing with varying dNTPs. In theory, there is a 75% probability a wrong base will pair with this ambiguous base, which will generate mutations in subsequent rounds of replication.
- Erroneous PCR. Taq DNA polymerase typically has lower-fidelity in comparison to other polymerases (e.g., Pfu and Vent). Furthermore, altering external conditions, such as buffer composition (e.g., high pH or high magnesium concentration), can affect the frequency of errors. Under various combinations of external conditions, the error rate of Taq PCR can be increased to 1 in 150 base pairs, from 1 in 633.
- Use of ambiguous base analogs (e.g., deoxyinosine). As already stated, deoxyinosine triphosphate (dI) can base pair with different dNTPs. In a reaction that contains dI and adjusted dNTPs concentrations (e.g., typical levels of three dNTPs and low levels of the fourth dNTP), a dI is likely to be incorporated into the newly synthesized sequence. Theoretically, there is a 75% chance that a wrong base will pair with a dI during subsequent replication events. This technique can result in 1 in 250 base pair mutation rates.
- Use of mutagenic agents. A classic method for causing widespread random mutations is by exposing a cell or organism to a mutagen (e.g., ENU).
Major applications of site directed mutagenesis are highlighted below:
- Site directed mutagenesis is used to study changes in protein activity that occurs as a result of the DNA manipulation.
- It is used to assess the activity of proteins containing known amino acid substitutions.
- It is used to select or screen for mutations (at the DNA, RNA or protein level) that have a desired property; and which can be manipulated further for enhanced biological activity.
- It is used to change specific amino acid in an enzyme. In this case, the modified enzyme molecule is assayed and compared to the wild-type enzyme molecule.
- It is used to introduce or remove restriction enzymes or endonuclease sites or tags.
- Site-directed mutagenesis helps scientists to study the mechanism of action behind biological reactions catalyzed by enzymes in vivo.
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