The authors would like to thank University of Southern Denmark open access policy grants.

1. Vector selection
<ol>
	<li>Choose a vector to perform downstream experiments with. Any vector is applicable for this 2- and 3-step PCR method.</li>
	<li>Based on choice of vector, choose appropriate restriction enzymes for cloning.</li>
</ol>
2. Primer design for site directed mutagenesis
<ol>
	<li>Decide between either the 2-step or 3-step PCR strategy (2-step is only for mutations &#60;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.</li>
	<li>Design primers for 2-step PCR.
	<ol>
		<li>Design primer 1 and 3 to amplify the DNA of interest and with a 5&#8217; overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.</li>
		<li>Design primer 2 to introduce mutation(s) at the desired site(s) and flank the mutation with 10-15 complementary nucleotides on both sides. Make the primer reverse if the mutation is introduced at the 5&#8217; end or forward if the mutation is introduced at the 3&#8217; end.</li>
	</ol>
	</li>
	<li>Design primers for 3-step PCR.
	<ol>
		<li>Design primer 1 and 4 to amplify the DNA of interest and with a 5&#8217; overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.</li>
		<li>Design primer 2 and 3 to introduce mutation(s) at desired site(s) and flank the mutation by 10-15 complementary nucleotides on both sides. Primer 2 and 3 are reverse complementary.</li>
	</ol>
	</li>
</ol>
3. PCR amplification of wild type DNA for cloning
NOTE: For details on PCR, see6.
<ol>
	<li>Perform PCR6 using primers 1+2 (2-step PCR) or 1+4 (3-step PCR) and use wild type DNA as template to obtain PCR product I. Use the PCR program in Table 1.</li>
	<li>Validate PCR by agarose gel electrophoresis.
	<ol>
		<li>Make an agarose gel solution (2%) by adding 2 g of agarose per 100 mL of 1x Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer. Dissolve the agarose by boiling in a microwave oven. Add the DNA-staining dye ethidium bromide (to a final concentration of ~ 0.5 &#181;g/mL) to the agarose gel solution for visualization.</li>
		<li>Cast agarose gel, place it in an electrophoresis unit, and load PCR samples (mixed with DNA loading dye) and a DNA ladder of known size. Run samples at 75 W for 45 min, or until bands are separated adequately, and visualize bands at an ultra-violet (UV) table or with a gel imaging system.</li>
	</ol>
	</li>
	<li>Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
	<li>Store the purified DNA at -20 &#176;C (in Tris-EDTA (TE) buffer &#8211; long term storage) or 4 &#176;C (in dH2O &#8211; short term storage) until used in step 5.</li>
</ol>
4. PCR to introduce site-directed mutations in the DNA
<ol>
	<li>PCR for 2-step PCR (see step 4.2 for 3-step PCR)
	<ol>
		<li>Perform PCR6 using primers 1+2 if mutations are in the 5&#8217; end or 2+3 if the mutations are in the 3&#8217; end and use wild type DNA as a template to obtain PCR product II (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used in step 5.</li>
		<li>Perform PCR6 using PCR product II as the primer together with primer 3 if mutations are in the 5&#8217; end or primer 1 if the mutations are in the 3&#8217; end and use wild type DNA as template to obtain PCR product III (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until step 5.</li>
	</ol>
	</li>
	<li>PCR for 3-step PCR
	<ol>
		<li>Perform PCR6 using primers 1+2 and 3+4 in separate reactions and use wild type DNA as template to obtain PCR product II and III (see Table 1 for PCR program).</li>
		<li>Validate PCRs by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify PCR products with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O).</li>
		<li>Perform PCR6 using primers 1+4 and use 2-5 ng of both PCR products II and III as the templates (in the same reaction) to obtain PCR product IV (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2. 
		NOTE: It is not unusual to get several incorrect bands (can sometimes be reduced by using less template). However, the incorrect PCR products can be ignored if the correctly sized band is excised and gel-extracted.</li>
		<li>If correct, purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until step 5.</li>
	</ol>
	</li>
</ol>
5. Recombination of wild type and mutant version(s) of DNA into the chosen vector
NOTE: For details on following steps, see7.
<ol>
	<li>Digest purified PCR products I and III (from 2-step PCR) and/or IV (from 3-step PCR) using the relevant restriction enzymes.
	<ol>
		<li>In 20 &#181;L of 1x digestion buffer with 1 &#181;L of each restriction enzyme, digest 200 ng of PCR product at 37 &#176;C for 30-60 min.</li>
	</ol>
	</li>
	<li>Purify digested PCR product by gel-extraction using a gel extraction kit (Table of Materials), measure the DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used for step 5.6.</li>
	<li>Digest purified vector with relevant restriction enzymes and treat with alkaline phosphatase to decrease vector re-ligation events. Do not treat PCR products with alkaline phosphatase.
	<ol>
		<li>In 30 &#181;L of 1x digestion buffer with 1 &#181;L of each restriction enzyme and 1 &#181;L of alkaline phosphatase, digest 1,000 ng of the vector at 37 &#176;C for 30-60 min.</li>
	</ol>
	</li>
	<li>Separate digested vector from waste DNA (e.g., uncut vector and cut-out DNA) using agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
	<li>Purify digested vector by gel-extraction using a gel extraction kit (Table of Materials), measure DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used for step 5.6.</li>
	<li>Ligate digested PCR products into digested vector with the reactions specified in Table 2.</li>
	<li>Incubate at room temperature for 2 h or overnight at 16 &#176;C.</li>
	<li>Transform recipient strain (e.g., E. coli K12) with ligation reactions.
	<ol>
		<li>Grow strain to OD600=0.3-0.5 and transfer a 1 mL culture to as many 1.5 mL tubes as ligase reactions.</li>
		<li>Spin at 3,500 x g for 5 min and discard supernatant.</li>
		<li>Resuspend cells in 200 &#956;L of transformation buffer (10 mL of lysogeny broth (LB) with 0.1 g/mL polyethylene glycol 3350, 5% dimethyl sulfate and 20 mM MgCl2).</li>
		<li>Add ligation reaction, and place the tubes on ice for 30 min.</li>
		<li>Heat-shock for 2 min at 42 &#176;C.</li>
		<li>Add 1 mL of LB to the 1.5 mL tubes, and allow phenotypic expression of antibiotic resistance for at least 45 min at 37 &#176;C.</li>
		<li>Spin the cells at 3,500 x g for 5 min, discard 1 mL of supernatant, and resuspend the cells in the remaining supernatant.</li>
		<li>Plate the cells on plates with appropriate antibiotics and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Identify transformants harboring vectors with successful integration of DNA insert (e.g., by PCR using vector- and insert-specific primers).</li>
	<li>Validate sequence of DNA by sanger sequencing. 
	CAUTION: Do not use the same primers for sequencing as used for step 5.9.</li>
</ol>
6. Using constructed vectors for in vitro and/or in vivo experiments
<ol>
	<li>In vivo experiment 
	NOTE: This is an example of using a vector to express wild type/mutated RNA to characterize post-transcriptional regulation. For further details on western blotting, see8.
	<ol>
		<li>Grow E. coli K12 strains with constructed vectors in appropriate medium and induce expression if required. Harvest samples by centrifugation.</li>
		<li>Prepare samples for sodium dodecyl sulfate&#8211;polyacrylamide gel electrophoresis (SDS-PAGE) by dissolving cell pellets in 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% bromophenol blue, 5% &#946;-mercaptoethanol, 10% glycerol) and boil at 95 &#176;C for 5 min. 
		NOTE: It is possible to cast a gel or use a commercially available precast gel. The latter was used in the results presented in Figure 3 (Table of Materials).</li>
		<li>Load 107 cells of each sample in separate wells (include a protein ladder), and run gel at 200 V until proteins are separated (approximately 45 min).</li>
		<li>Blot the proteins onto a cellulose-membrane by semi-dry transfer at 80 mA for 1 h.</li>
		<li>Block the membrane with a mixture of proteins (e.g., 5% milk-powder dissolved in 1x Tween 20-Tris-buffered saline (TTBS) buffer).</li>
		<li>Add primary antibody (dissolved in 1x TTBS buffer) that target the protein of interest (e.g., GFP-, FLAG-, or HIS-tagged protein) and incubate for 1 h with gentle agitation.</li>
		<li>Wash membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.</li>
		<li>Add secondary antibody (dissolved in 1x TTBS buffer) that targets the primary antibodies and allow for detection (e.g., horseradish peroxidase (HRP)-conjugated secondary antibodies. Incubate for 1 h with gentle agitation.</li>
		<li>Wash the membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.</li>
		<li>Visualize the membrane with a technique compatible with the secondary antibodies (e.g., by imaging after incubation with a luminol-derived chemiluminescence, if a HRP-conjugated secondary antibody was used).</li>
	</ol>
	</li>
	<li>In vitro experiment 
	NOTE: This is an example of using the vector as a template for in vitro transcription of RNA to characterize RNA-protein interactions. For further details on EMSA, see9.
	<ol>
		<li>Make in vitro transcripts using a T7 in vitro transcription kit (Table of Materials) and vectors from step 5 as templates.</li>
		<li>Separate RNA transcripts by PAGE on a 4.5% 7 M urea denaturing gel, and extract RNA directly from the gel by electro elution with dialysis tubes (Table of Materials).</li>
		<li>Label RNA (e.g., radiolabeling with &#947;-32P-ATP using T4-polynucleotide kinase (Table of Materials) and purify again with columns (Table of Materials). 
		CAUTION: Before working with radioactive material, consult with the local radiation safety officer.</li>
		<li>Mix labelled-RNA with increasing concentrations of protein in separate reactions in a 1x binding buffer (20 mM Tris, pH 8, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT)). 
		NOTE: In the presented results (Figure 4), 2 nM of radiolabeled csgD mRNA was mixed with a gradient of 0 to 2 &#181;M Hfq protein (monomer concentration).</li>
		<li>Allow protein and RNA to hybridize before loading hybridization mix on a non-denaturing polyacrylamide gel and run the gel for 1.5 h at 200 V.</li>
		<li>Visualize the gel with a technique compatible with the labeling from step 6.1.3. (e.g., by phosphoimaging if radiolabeling was applied).</li>
		<li>Quantify the relative intensity of the shifted bands with an imaging processing program and fit a curve (sigmoidal) to the data by using a graph and data analysis software (Table of Materials). Based on the fitted curve, dissociation constant (Kd) values can be determined automatically with the software.</li>
	</ol>
	</li>
</ol>

<ol><li>Bouvier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+RNA+binding+to+5%27+mRNA+coding+region+inhibits+translational+initiation%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Small RNA binding to 5' mRNA coding region inhibits translational initiation.</a> Molecular Cell. 32 (6), 827-837 (2008).</li>
<li>Jorgensen, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+regulatory+RNAs+control+the+multi-cellular+adhesive+lifestyle+of+Escherichia+coli%5BTitle%5D)+AND+%22Molecular+Microbioly%22%5BJournal%5D)">Small regulatory RNAs control the multi-cellular adhesive lifestyle of Escherichia coli.</a> Molecular Microbioly. 84 (1), 36-50 (2012).</li>
<li>Antal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+small+bacterial+RNA+regulates+a+putative+ABC+transporter%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">A small bacterial RNA regulates a putative ABC transporter.</a> The Journal of Biological Chemistry. 280 (9), 7901-7908 (2005).</li>
<li>Andreassen, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((sRNA-dependent+control+of+curli+biosynthesis+in+Escherichia+coli%3A+McaS+directs+endonucleolytic+cleavage+of+csgD+mRNA%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">sRNA-dependent control of curli biosynthesis in Escherichia coli: McaS directs endonucleolytic cleavage of csgD mRNA.</a> Nucleic Acids Research. , (2018).</li>
<li>Thomason, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+small+RNA+that+regulates+motility+and+biofilm+formation+in+response+to+changes+in+nutrient+availability+in+Escherichia+coli%5BTitle%5D)+AND+%22Molecular+Microbioly%22%5BJournal%5D)">A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli.</a> Molecular Microbioly. 84 (1), 36-50 (2012).</li>
<li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymerase+chain+reaction%3A+basic+protocol+plus+troubleshooting+and+optimization+strategies%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998(2012).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> Journal of Visualized Experiments. , Cambridge, MA. (2018).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+The+Western+Blot%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot.</a> Journal of Visualized Experiments. , Cambridge, MA. (2018).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((JoVE+Science+Education+Database.+Biochemistry.+Electrophoretic+Mobility+Shift+Assay+%28EMSA%29%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">JoVE Science Education Database. Biochemistry. Electrophoretic Mobility Shift Assay (EMSA).</a> Journal of Visualized Experiments. , Cambridge, MA. (2018).</li>
<li>Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+efficient+site-specific+mutagenesis+without+phenotypic+selection%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Rapid and efficient site-specific mutagenesis without phenotypic selection.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (2), 488-492 (1985).</li>
<li>Laible, M., Boonrod, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Homemade+site+directed+mutagenesis+of+whole+plasmids%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Homemade site directed mutagenesis of whole plasmids.</a> Journal of Visualized Experiments. (27), (2009).</li>
<li>Wells, J. A., Vasser, M., Powers, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cassette+mutagenesis%3A+an+efficient+method+for+generation+of+multiple+mutations+at+defined+sites%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.</a> Gene. 34 (2-3), 315-323 (1985).</li>
<li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+genome+engineering+using+CRISPR%2FCas+systems%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li>
<li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-guided+human+genome+engineering+via+Cas9%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li>
</ol>

The authors declare no competing interests.

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 ...

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 significantly, some mutations in the DNA can have huge implications. Thus, site-directed mutagenesis is a valuable tool for studying the importance of specific DNA sites at all levels.
This protocol describes a targeted mutagenesis approach used to introduce specific mutations. The protocol relies on two different PCR strategies: a 2-step or a 3-step PCR. The 2-step PCR is applicable if the desired mutation is close to either the 5&#39; end or the 3&#39; end of the DNA of interest (&#60;200 base pairs (bp) from the end) and the 3-step PCR is applicable in all cases.
In the 2-step PCR approach, 3 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 3, forward and reverse, respectively), and a single primer is designed to incorporate the mutation. The mutation introducing primer (primer 2) should have a reverse orientation if the mutation is close to the 5&#39; end and a forward orientation if the mutation is close to the 3&#39; end. In the first PCR step, primer 1+2 or 2+3 amplifies a small fragment close to the 5&#39; end or 3&#39; end, respectively. The resulting PCR product is then used as a primer in step two with primer 1 or 3, thus resulting in a PCR product with a mutation in the DNA of interest (Figure 1A).
In the 3-step PCR, 4 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 4, forward and reverse, respectively) and one set of primers is designed to incorporate specific mutations with overlapping complementarity (primers 2 and 3, reverse and forward, respectively). In step one and two, primers 1+2 and 3+4 amplify the 5&#39; and 3&#39; end. In step three, the resulting PCR products from step one and two are used as templates and amplified with primers 1+4. Thus, the resulting PCR product is the DNA of interest with the desired mutation (Figure 1B).
While the mutated DNA can be used for any downstream application, this protocol describes how to re-combine the DNA into a cloning vector. The use of cloning vectors has several advantages such as ease of cloning and specific experimental applications depending on features of the vector. This feature is often used for RNA interaction studies. Another technique for RNA interaction studies is structural probing of the RNA in complex with another RNA1,2 or protein3,4. However, structural probing is only performed in vitro whereas site-directed mutagenesis and subsequent cloning allow for interaction studies in vivo.
Site-directed mutagenesis has been extensively used for RNA interaction studies as presented here. However, the key method regarding 2- or 3-step PCR is applicable to any piece of DNA, and thus not only limited to RNA-interaction studies.
To exemplify the technique and its possible uses, characterization of regions important for post-transcriptional regulation of the mRNA, csgD, of Escherichia coli (E. coli) is used. In E. coli, csgD is targeted by the small non-coding RNA, McaS, in cooperation with a protein, Hfq, to repress protein-expression of CsgD2,4,5. The technique is used to introduce mutations to the base-pairing region between csgD and McaS, and to the Hfq binding site of csgD. The obtained DNA is then cloned into a vector suitable for subsequent experiments. Downstream applications of the technique include both in vivo and in vitro experiments. For illustration, example 1 is characterized in vivo using a western blot assay and example 2 is characterized in vitro using an electrophoretic mobility shift assay (EMSA). In both cases, it is illustrated how site-directed mutagenesis can be used in combination with other techniques to make biological conclusions about a gene of interest.

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

site-directed mutagenesis for in vitro and in vivo experiments exemplified with rna interactions in escherichia coli

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).

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 &#946;-D-1-thiogalactopyranoside (IPTG) inducible low copy plasmid with ampicillin resis...

Watch this Scientific Journal Video about Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli at JoVE.com

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

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.

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

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

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Research

JoVE Journal

Biochemistry

20.0K Views.  University of Southern Denmark. 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.

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

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetics

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Biology

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

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

Summary

Explore More Videos

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli