Site-directed mutagenesis is the deliberate introduction of a specified sequence alteration at a selected position or region of a DNA molecule. The intended alteration is encoded in a designed nucleic acid reagent and incorporated into newly synthesized or reconstructed DNA. Changes can include individual nucleotide substitutions, short insertions or deletions, and replacement of defined sequence segments [1][2]. In common polymerase-based formats, mutation-bearing primers anneal to a DNA template and direct synthesis of molecules containing the designed sequence. Primer complementarity, template sequence composition, polymerase properties, and subsequent processing influence the composition of the resulting DNA population [3]. The molecular endpoint is a DNA construct carrying the intended change within an otherwise specified sequence context [1][3][4]. Sequence analysis establishes the identity of that construct and detects unintended alterations that may accompany its generation [1][3].
- targeted mutation
- DNA primers
- sequence substitution
- DNA polymerase
References
- Xiang‐Jiao Yang; Seamless and Highly Efficient Site‐directed Mutagenesis for Protein, RNA, and Plasmid Engineering. Curr. Protoc. 2026, 6, e70303, 10.1002/cpz1.70303.
- Seungjin Kim; Seungwon Lee; Hyun Gyu Lim; Recent Advances in Targeted Mutagenesis to Expedite the Evolution of Biological Systems. J. Microbiol. 2025, 63, e2501008, 10.71150/jm.2501008.
- Paulina Varela-Castillo; Arezousadat Razavi; Negar Mousavi; Nicole Robinson; Xiang-Jiao Yang; Efficiency and Fidelity of Site-Directed Mutagenesis with Complementary Primer Pairs. Cells 2026, 15, 138, 10.3390/cells15020138.
- T A Kunkel; Rapid and Efficient Site-specific Mutagenesis Without Phenotypic Selection.. Proc. Natl. Acad. Sci. 1985, 82, 488-492, 10.1073/pnas.82.2.488.