Question

Which technique is not used for site directed mutagenesis?

• A. Oligonucleotide directed mutagenesis
• B. Cassette mutagenesis
• C. Physical mutagenesis
• D. SDM with PCR

Hint:

• Site-Directed Mutagenesis (SDM) Introduces specific, desired mutations at a defined location in a DNA sequence.
• Common SDM techniques: Oligonucleotide-directed (e.g., Kunkel method), PCR-based methods, cassette mutagenesis.
• Random Mutagenesis Introduces mutations non-specifically throughout a gene or genome. Can be induced by chemical mutagens (e.g., EMS, NTG) or physical mutagens (e.g., UV, X-rays).

Answer 1

The Correct Option is C

Site-directed mutagenesis (SDM) is a molecular biology technique used to create specific, targeted changes (mutations) in a DNA sequence. The goal is to alter one or more specific nucleotides at a defined site within a gene or DNA fragment. Common techniques for site-directed mutagenesis include:

• (a) Oligonucleotide-directed mutagenesis: This is a classic and widely used method. It involves using a short, synthetic DNA oligonucleotide (primer) that contains the desired mutation. This primer is annealed to a single-stranded DNA template (e.g., from a phagemid or denatured plasmid), and DNA polymerase extends it to create a complementary strand incorporating the mutation. Subsequent replication and selection yield DNA molecules with the specific mutation.
• (b) Cassette mutagenesis: Involves synthesizing a DNA fragment (cassette) containing the desired mutation(s) and then ligating this cassette into a target DNA molecule by replacing a corresponding wild-type segment between two restriction enzyme sites.
• (d) SDM with PCR (PCR-based site-directed mutagenesis): Various PCR-based strategies are used for SDM, often involving primers that contain mismatches to introduce the desired mutation. Examples include overlap extension PCR, megaprimer PCR, and QuikChange site-directed mutagenesis.

Let's analyze the options: (a), (b), and (d) are all established techniques for site-directed mutagenesis, aiming for specific changes at defined locations. (c) Physical mutagenesis: This refers to the use of physical mutagens (e.g., UV radiation, X-rays, ionizing radiation) to induce mutations in DNA. Physical mutagens generally cause random mutations throughout the genome, rather than targeted changes at a specific site. While physical mutagenesis is used to create mutants for screening or research, it is not a technique for site-directed mutagenesis, which implies precision and control over the location and type of mutation. Therefore, physical mutagenesis is not used for site-directed mutagenesis. \[ \boxed{\text{Physical mutagenesis}} \]