Publikationer pr. år
Publikationer pr. år
Patrick Rosendahl Andreassen, Jens Sivkær Pettersen, Mikkel Jørgensen
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).
Originalsprog | Engelsk |
---|---|
Artikelnummer | e58996 |
Tidsskrift | Journal of visualized experiments : JoVE |
Udgave nummer | 144 |
ISSN | 1940-087X |
DOI | |
Status | Udgivet - 5. feb. 2019 |
Publikation: Afhandling › Ph.d.-afhandling