In this article, I briefly describe mutagenesis and mutagenesis of cloned genes.
Mutagenesis
Mutagenesis is a process in which an organism changes its DNA. It results in gene mutation. Mutagenesis can be spontaneous or induced. Specified mutations in genes can be engineered and the effects of these mutations can be tasted. The availability of cloned DNA sequences for genes makes this possible. The two basic strategies for performing cloned DNA mutagenesis are random and site-directed mutagenesis (mutagenesis in vitro).
In random mutagenesis, mutations are induced in the cloned DNA, and the mutagenized cloned DNA is tested for a specific function. In site-directed mutagenesis, single nucleotide changes are introduced at specific positions and are used for fine mapping of genetic functions.
Deletion mutagenesis
To define the importance of particular sequences, deleting DNA from one end is quite useful. In cDNA clones, deletion from the ends of the coding region produces either N-terminally or C-terminally truncated proteins. The N-terminal domain of a given protein could be a DNA-binding domain. The central region is an ATP binding site and the C-terminal region could help the protein to interact to form dimers.
In genomic clones, after the identification of transcription start sites, upstream sequences are progressively removed. It is done to discover the minimum length of the upstream sequence that has promoter and regulatory function. The enzyme exonuclease III creates unidirectional deletions by removing one strand in a 3′ to 5′ direction from a recessed 3′ end. A single strand-specific nuclease then creates blunt end molecules for ligation, and transformation generates the deleted clones.
Site-directed mutagenesis
When a mutation is created at a particular site in a DNA sequence of a gene, it is known as site-directed mutagenesis. The process involves the synthesis of a short DNA primer which is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. This synthetic primer is complementary to the template DNA around the base change so it can hybridize with the DNA containing the gene of interest.
A DNA polymerase extends the single-stranded primer and copies the rest of the gene. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene (figure 1). The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected by DNA sequencing. This is done to check whether the mutants contain the desired mutation.
Oligonucleotide-based method
Site-directed mutagenesis has been widely used in the study of protein functions. Among many approaches, Michael Smith developed an oligonucleotide-based method. He was awarded a Nobel Prize for his discovery. This method involves cloning the DNA of interest into a plasmid vector.
The plasmid vector is first denatured to produce single strands. Then, a synthetic oligonucleotide with the desired mutation is annealed to the target region. The mutant oligonucleotide is then extended using a plasmid DNA strand as the template. After that, the heteroduplex is propagated by transformation in E.coli (figure 1). Theoretically, about 50% of the produced heteroduplexes will be mutants and the other 50% will be the “wild type” (without mutation) after propagation.
The original method using single-primer extension gives a lower yield of mutants. The resulting mixture may contain both the original un-mutated template as well as the mutant strand. Thus, it produces a mixed population of mutant and non-mutant progenies. The mutants may also be counter-selected due to the presence of a mismatch repair system, which favors the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.
PCR site-directed mutagenesis
When PCR is used to make changes in the DNA nucleotide sequence, this is called PCR mutagenesis. This method can be utilized to assess the function of a promoter and to alter amino acids to test the functions of domains in a protein.
There are various methods through which mutations can be introduced. A pair of primers are designed that have altered sequences and which overlap by at least 20 nt. In a standard vector pGEM, SP6 and T7 are two standard primer-binding sites that can be used in combination with the mutagenic primers.
Two separate PCR reactions are performed. One of which amplifies the 5′ portion of the insert using SP6 and the reverse primer, and the other amplifies the 3′ portion of the insert using the forward and T7 primers. If the two PCR products are purified, mixed, and amplified using SP6 and T7 primers, then the product comes out as a full-length, mutated molecule. This method is nearly 100% efficient and very quick. An alternative and more convenient version involves using the forward and reverse mutagenic primers to extend around the plasmid containing the target gene (figure 2).
At the end of the reaction, some of the products consist of circular molecules with the mutation in both strands and nicks at either end of the primer sequence. These circular molecules do not need restriction digestion and ligation and can be transformed directly to give clones containing the mutant plasmid. All mutants undergo sequencing to be checked before use. Instead of using the whole of the PCR-generated DNA, a smaller fragment containing the mutated region could be sub-cloned into the equivalent sites in the original clone.
Conclusion
The two basic strategies for performing cloned DNA mutagenesis are random and site-directed mutagenesis (mutagenesis in vitro). Single nucleotide changes are introduced at specific positions in site-directed mutagenesis and are used for fine mapping of genetic functions.
In cDNA clones, deletion from the ends of the coding region produces either N-terminally or C-terminally truncated proteins. In genomic clones, after the identification of transcription start sites, upstream sequences are progressively removed. It is done to discover the minimum length of the upstream sequence that has promoter and regulatory function.
Site-directed mutagenesis results, when a mutation is created at a particular site in a DNA sequence of a gene. The process involves the synthesis of a short DNA primer. The short primer is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. When PCR is used to make changes in the DNA nucleotide sequence, this is called PCR mutagenesis. This method can be utilized to assess the function of a promoter and to alter amino acids to test the functions of domains in a protein.
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I, Swagatika Sahu (author of this website), have done my master’s in Biotechnology. I have around twelve years of experience in writing and believe that writing is a great way to share knowledge. I hope the articles on the website will help users in enhancing their intellect in Biotechnology.