Abstract
Site-directed mutagenesis (SDM) is a technique in molecular biology and protein engineering that is widely used to determine the significance of specific residues involved in post-translational modifications (PTMs), protein structure, function, and stability. Here, we describe a simple and cost-effective polymerase chain reaction (PCR)-based SDM method. This method can be used to introduce point mutation, short addition, or deletions in protein sequences. Using polycomb repressive complex-2 (PRC2)-associated protein JARID2 as an example, we demonstrate how SDM can be used to study structural and consequently functional changes in a protein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Edelheit O, Hanukoglu A, Hanukoglu I (2009) Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol 9:61. https://doi.org/10.1186/1472-6750-9-61
Silva D, Santos G, Barroca M, Collins T (2017) Inverse PCR for point mutation introduction. Methods Mol Biol 1620:87–100. https://doi.org/10.1007/978-1-4939-7060-5_5
Zhang K, Yin X, Shi K, Zhang S, Wang J, Zhao S, Deng H, Zhang C, Wu Z, Li Y, Zhou X, Deng W (2021) A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Sci Rep 11(1):10454. https://doi.org/10.1038/s41598-021-89884-z
Braman J, Papworth C, Greener A (1996) Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol 57:31–44. https://doi.org/10.1385/0-89603-332-5:31
Lu H, Li G, Zhou C, Jin W, Qian X, Wang Z, Pan H, Jin H, Wang X (2016) Regulation and role of post-translational modifications of enhancer of zeste homologue 2 in cancer development. Am J Cancer Res 6(12):2737–2754
Yang Y, Li G (2020) Post-translational modifications of PRC2: signals directing its activity. Epigenetics Chromatin 13(1):47. https://doi.org/10.1186/s13072-020-00369-1
Al-Raawi D, Jones R, Wijesinghe S, Halsall J, Petric M, Roberts S, Hotchin NA, Kanhere A (2019) A novel form of JARID2 is required for differentiation in lineage-committed cells. EMBO J 38(3). https://doi.org/10.15252/embj.201798449
Kasinath V, Faini M, Poepsel S, Reif D, Feng XA, Stjepanovic G, Aebersold R, Nogales E (2018) Structures of human PRC2 with its cofactors AEBP2 and JARID2. Science 359:940–944. https://doi.org/10.1126/science.aar5700
Acknowledgments
This work was supported by University of Liverpool and Islamic Development Bank.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Al-Raawi, D., Kanhere, A. (2023). Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function. In: Lanzuolo, C., Marasca, F. (eds) Polycomb Group Proteins. Methods in Molecular Biology, vol 2655. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3143-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3143-0_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3142-3
Online ISBN: 978-1-0716-3143-0
eBook Packages: Springer Protocols