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A Brief Overview on Mutagenesis:


Introduction:

Mutation is often confused with the process of mutation. Mutagenesis occurs when an organism’s DNA undergoes a change either experimentally or naturally which leads to mutation. It has said to be a force of evolution but can also be critical.

Process of Mutagenesis:

Mutagenesis is used in molecular biology techniques in order to create unique mutant genes, organisms and proteins. The following are the primary methods or techniques of mutagenesis:



Site targeted mutagenesis: It is a technique in which DNA can be modified at a specific nucleotide location, resulting in a predetermined change in amino acid. These substitution mutations can result in significant changes in protein conformation and function. These techniques involve (1) a DNA template with a target gene, (2) knowledge of the nucleotide sequence of the target gene, and (3) a short primer (mainly 20 to 30 base pairs) complementary to the target sequence that is modified to incorporate an inaccurate nucleotide.



Random and excessive mutagenesis: This is a viable technique when several mutations are desired; however, there is less control over the resulting mutations. This technique has helped to map primary protein sequences (e.g., EcoRV restriction endonuclease). It has various methods such as:


  • Primers randomly produced with mismatched bases: 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.


Erroneous PCR: Taq DNA polymerase typically has lower-fidelity in comparison to other polymerases (e.g., Pfu and Vent) altering external conditions, such as high pH or high magnesium concentration, can affect the frequency of errors.

  • Use of ambiguous base analogs: Deoxyinosine triphosphate (dI) can also be used as base pair for various dNTPs. In a reaction that includes dI and modified amounts of dNTPs (e.g. normal levels of three dNTPs and low levels of the fourth dNTP), dI is likely to be integrated into the freshly synthesized chain. Theoretically, there is a 75% possibility that an incorrect base will be paired with a dI during subsequent replication cases. This procedure may result in a mutation rate of 1 in 250 base pairs.

  • Use of mutagenic agents: The conventional mechanism used to induce widespread random mutations is by exposing the cell or organism to a mutagen e.g., ENU.


CRISPR-Cas 9 is another technique in accomplishing the process of mutagenesis. This technology is a genome-editing tool derived from bacterial defenses against viruses and foreign plasmids. It has two vital components: a guide RNA (gRNA) that binds complementary to a target DNA sequence, and an endonuclease (Cas9) Cas9-induced site-specific dsDNA breaks induce cell repair mechanisms, which can be exploited to modify DNA.


Clinical importance of mutagenesis:

Carcinogenesis is when a cell begins to divide uncontrollably. Mutations of oncogenes (which promote cell growth) can generate a clonal cell population with highly proliferative properties. Two-thirds of cancer driver mutations are attributable to spontaneous mutagenesis.


Heritable diseases emerge from mutagenesis, as irreversible DNA modifications are passed over to the descendants. Sickle cell anemia is an autosomal recessive disorder induced by a missense mutation in the B-globin gene. Research shows that this mutation has created an adaptive advantage for heterozygous transporters in malaria-endemic areas. Homozygous inheritance has a poor prognosis enhanced danger of anemia, infection, and stroke and organ damage.


Precision Medicine is a term in which disease therapy is centered on the knowledge of individual genome variations that have become more important as whole genome sequences have become more available. It relies on innovative clinical techniques, drug discovery and gene-oriented therapy. Gene-targeted techniques such as CRISPR/Cas9 and TALENs will be used clinically in near future. CRISPR is being tested for the treatment of single-gene mutations (e.g., DMD, cystic fibrosis), HIV and cancers.



Recent research work in mutagenesis:

Adenosine Deaminases are enzymes that act on the RNA editing. Diseases which are associated with this enzyme are still not known. High throughput mutagenesis has been studied to know the specific residues of ADARAs.


REFERENCES:

1. Hryhorowicz M, Lipiński D, Zeyland J, Słomski R. CRISPR/Cas9 Immune System as a Tool for Genome Engineering. Arch Immunol Ther Exp (Warsz). 2017 Jun;65(3):233-240. [PMC free article: PMC5434172] [PubMed: 27699445]


2. Ayatollahi H, Keramati MR, Shirdel A, Kooshyar MM, Raiszadeh M, Shakeri S, Sadeghian MH. BCR-ABL fusion genes and laboratory findings in patients with chronic myeloid leukemia in northeast Iran. Caspian J Intern Med. 2018 Winter;9(1):65-70. [PMC free article: PMC5771363] [PubMed: 29387322]


3. Ling MM, Robinson BH. Approaches to DNA mutagenesis: an overview. Anal Biochem. 1997 Dec 15;254(2):157-78. [PubMed: 9417773]

Durland J, Ahmadian-Moghadam H. Genetics, Mutagenesis. [Updated 2020 Sep 20]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK560519/


4. Park S, Doherty EE, Xie Y, Padyana AK, Fang F, Zhang Y, Karki A, Lebrilla CB, Siegel JB, Beal PA. High-throughput mutagenesis reveals unique structural features of human ADAR1. Nature communications. 2020 Oct 12;11(1):1-3.


Blog Author: Naushin Raheema

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