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 PC...

<ol>
	<li>Bouvier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA+binding+to+5'+mRNA+coding+region+inhibits+translational+initiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+regulatory+RNAs+control+the+multi-cellular+adhesive+lifestyle+of+Escherichia+coli.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bacterial+RNA+regulates+a+putative+ABC+transporter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sRNA-dependent+control+of+curli+biosynthesis+in+Escherichia+coli:+McaS+directs+endonucleolytic+cleavage+of+csgD+mRNA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+RNA+that+regulates+motility+and+biofilm+formation+in+response+to+changes+in+nutrient+availability+in+Escherichia+coli.">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. 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Molecular Cloning.</a> Journal of Visualized Experiments. , (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+The+Western+Blot.">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot.</a> Journal of Visualized Experiments. , (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Biochemistry.+Electrophoretic+Mobility+Shift+Assay+(EMSA).">JoVE Science Education Database. Biochemistry. Electrophoretic Mobility Shift Assay (EMSA).</a> Journal of Visualized Experiments. , (2018).</li><li>Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+site-specific+mutagenesis+without+phenotypic+selection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homemade+site+directed+mutagenesis+of+whole+plasmids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassette+mutagenesis:+an+efficient+method+for+generation+of+multiple+mutations+at+defined+sites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li></ol>

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 ...

Site-directed mutagenesis is useful to study molecular interactions, such as RNA-RNA, and RNA protein interactions. This protocol describes how to perform site-directed mutagenesis with a two or 3-step PCR approach. The main advantages of the method are the diversity of different mutations that can be incorporated, as well as a relative ease and cost for doing so.To begin, use the wild-type DNA template and primer pairs two, one, and two, three, specific for 2-step, or primer pairs three, one, and three, four, specific for 3-step PCR to obtain PCR product one. To validate PCR products by agarose gel electrophoresis, make a 2%agarose gel by dissolving two grams of agarose in 100 milliliters of 1X TAE buffer, using a microwave oven. Add ethidium bromide at a final concentration of 0.5 microgram per milliliter.Then cast the agarose gel. When solidified, place it in an electrophoresis unit. Load a DNA ladder of known size, and the PCR samples mixed with DNA loading dye into wells.Run samples at 75 volts for 45 minutes. And visualize bands on a UV transilluminator or with a gel imaging system. Next, purify the PCR product with a gel extraction kit according to the manufacturers protocol and measure the concentration of the purified DNA with a spectrophotometer.Then store the purified DNA at minus 20 celsius in TE buffer. To begin site-directed mutagenesis with a 2-step PCR, first use the wild-type DNA template, then the primer pairs two, one and two, two, for five primer mutations, or primer pairs two, two, and two, three for three priment mutations to obtain PCR product two. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.Then use the wild-type DNA template and the PCR product two as the primer, together with primer two, three for five priment mutation, or primer two, one for three priment mutation, to obtain PCR product three. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Then store the purified DNA at minus 20 degrees celsius in TE buffer.To begin site-direct mutagenesis with a 3-step PCR, first use the wild-type DNA template and primer pairs three, one, and three, two to obtain PCR product two. Use the wild-type DNA template and primer pairs three, three, and three, four, to obtain PCR product three. Use the wild-type DNA template and primer pairs three, three and three, four, to obtain PCR product three.Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Finally use two to five nanograms of PCR products two and three as the template along with primer pairs three, one and three, four, to obtain PCR product four. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.Store the purified DNA at minus 20 degrees celsius in TE buffer. In this study, 2-step and 3-step site-directed mutagenesis were used to investigate RNA interactions regarding post transcriptional regulation of CsgD. The wild-type CsgD was PCR amplified to yield PCR product IA, and the wild-type mcaS was PCR amplified to yield PCR product IB.Using a 3-step PCR strategy, PCR products IIA and IIIA were synthesized in the first two steps, and IVA in the third step to introduce mutations in CsgD.Similarly, complimentary site-directed mutations were introduced in mcaS to yield PCR products IIB, IIIB, in the first two steps, and IVB in the third step. Western blot analysis showed that while expression of wild-type mcaS prevents translation of wild-type CsgD, mutations in either mcaS or CsgD alleviates the observed repression. However, mutations in both CsgD and mcaS prevent translation of CsgD.Using a 2-step PCR strategy, PCR products IIC, IID, IIE, IIF, and IIG, were synthesized in the first step, and PCR products IIIC, IIID, IIIE, IIIF, and IIIG, in the second step to introduce mutations to the entire CsgD DNA. EMSA results of the HFQ binding to in vitro transcribed CsgD wild-type and mutant RNAs, synthesized using PCR products IIIC, IIID, IIIE, IIIF, and IIIG, showed the increased KD values of the mutant alleles. This proved that HFQ could bind less efficiently to the mutants.Furthermore HFQ has several binding sites on CsgD, which is shown by the three shifts observed for the wild-type RNA. However, only two binding sites are observed for different mutant RNAs. Thus the site-directed mutational approach identifies primary and or secondary structures of the CsgD mRNA that are important for complete binding of HFQ.The method for site-directed mutagenesis described here relies on PCR. Good PCR practices are therefor crucial for the method and might require optimization to be successful. Site-directed mutagenesis is only powerful with all three methods such EMSA and Western Blotting.Other methods, for instance, include various structural assays and some activity assays, and post translation modification studies.

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Biochemistry

19.5K vistas.  University of Southern Denmark. La mutagénesis dirigida por el sitio es útil para estudiar las interacciones moleculares, como el ARN-ARN, y las interacciones de proteínas de ARN. Este protocolo describe cómo realizar la mutagénesis dirigida por el sitio con un enfoque de PCR de dos o 3 pasos. Las principales ventajas del método son la diversidad de diferentes mutaciones que se pueden incorporar, así como una relativa facilidad y costo para hacerlo.Para empezar, utilice la plantilla de ADN de tipo salvaje y lo...