Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Summary

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Abstract

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).

Introduction

DNA is often referred to as the blueprint of a living cell since all structures of the cell are encoded in the sequence of its DNA. Accurate replication and DNA repair mechanisms ensure that only very low rates of mutations occur, which is essential for sustaining correct functions of coded genes. Changes of the DNA sequence can affect successive functions at different levels starting with DNA (recognition by transcription factors and restriction enzymes), then RNA (base-pair complementarity and secondary structure alterations) and/or protein (amino acid substitutions, deletions, additions or frame-shifts). While many mutations do not affect gene function significantl....

Protocol

1. Vector selection

1. Choose a vector to perform downstream experiments with. Any vector is applicable for this 2- and 3-step PCR method.
2. Based on choice of vector, choose appropriate restriction enzymes for cloning.

2. Primer design for site directed mutagenesis

1. Decide between either the 2-step or 3-step PCR strategy (2-step is only for mutations <200 bp from either end of the DNA of interest). For the 2-step PCR, go to step 2.2 and for the 3-step PCR go to step 2.3.
2. Design primers for 2-step PCR.
   1. Design primer 1 and 3 to amplify the DNA of interest and with a 5’ overha....

Results

To investigate RNA interactions regarding post-transcriptional regulation of csgD, a double vector setup was chosen: one to express the csgD mRNA and another to express the small non-coding RNA, McaS. csgD was cloned into pBAD33, which is an arabinose inducible medium-copy plasmid with chloramphenicol resistance and McaS was cloned into mini R1 pNDM220, which is an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible low copy plasmid with ampicillin resis.......

Discussion

Site-directed mutagenesis has a broad array of different applications, and here, representative results from an in vivo and an in vitro experiment were included as examples of how to make biological conclusions using the technique. Site-directed mutagenesis has for long been the golden standard for RNA interaction studies. The strength of the technique lies in the combination of introducing relevant mutations with downstream assays and experiments (e.g., western blot or EMSA) to draw conclusions about specific DNA sites .......

Materials

Name	Company	Catalog Number	Comments
Anti-GroEL antibody produced in rabbit	Merck	G6532	Primary antibody
Azure c200	Azure	NA	Gel imaging workstation
Custom DNA oligo	Merck	VC00021
DeNovix DS-11	DeNovix	NA	Spectrophotometer for nucleic acid measurements
DNA Gel Loading Dye (6X)	Thermo Scientific	R0611
Ethidium bromide solution 1 %	Carl Roth	2218.1
GeneJET Gel Extraction Kit	Thermo Scientific	K0691
GeneRuler DNA Ladder Mix	Fermentas	SM0333
Gerard GeBAflex-tube Midi	Gerard Biotech	TO12	Dialysis tubes for electro elution
MEGAscript T7 Transcription Kit	Invitrogen	AM1334
Mini-Sub Cell GT Cell	Bio-Rad	1704406	Horizontal electrophoresis system
Monoclonal ANTI-FLAG M2 antibody produced in mouse	Merck	F3165	Primary antibody
Mouse Immunoglobulins	Dako Cytomation	P0447	HRP conjucated secondary antibody
NucleoSpin miRNA	Macherey Nagel	740971	RNA purification
NuPAGE 4-12% Bis-Tris Protein Gels	Thermo Scientific	NP0323BOX	Bis-Tris gels for protein separation
Phusion High-Fidelity PCR Master Mix with HF Buffer	New England Biolabs	M0531S	DNA polymerase
PowerPac HC High-Current Power Supply	Bio-Rad	1645052
Rabbit Immunoglobulins	Dako Cytomation	P0448	HRP conjucated secondary antibody
SeaKem LE Agarose	Lonza	50004
SigmaPlot	Systat Software Inc	NA	Graph and data analysis software tool
T100 Thermal Cycler	Bio-Rad	1861096	PCR machine
T4 DNA ligase	New England Biolabs	M0202	Ligase
T4 Polynucleotide Kinase	New England Biolabs	M0201S
TAE Buffer (Tris-acetate-EDTA) (50X)	Thermo Scientific	B49