This project was partially funded by 60% Ateneo grants from Ministero dell&#39;Universit&#224;, Italy to S.M., and E.T. We also received partial contributions to research expenses by the Department of Medical, Oral and Biotechnological Sciences, University of Chieti &#34;G. d&#39;Annunzio&#34;.

The protocol follows the guidelines of the Medical Research Ethics Committee of the University of Chieti.
1. Reagent Setup
<ol>
	<li>Prepare the Tris-EDTA (TE) buffer (pH 8.0). Add 200 &#181;L of EDTA 0.5 M and 1 mL of Tris-Cl 1 M in a beaker and bring to 100 mL with sterile water. TE buffer final concentration: 10 mM Tris-Cl, 1 mM EDTA. Store at room temperature (RT).</li>
	<li>Prepare 1 L of the 10x stock solution electrophoresis buffer (TBE). Dissolve 108 g of Tris Base, 55 g of boric acid, and 40 mL of EDTA 0.5 M (pH 8) into a beaker, and bring to volume of 1 L with sterile water. Store at RT.</li>
	<li>Prepare the gel loading dye. Dissolve 0.25 g of bromophenol blue, 0.25 g of xylene cyanol FF, 50 g of glycerol, 1 mM of EDTA (pH 8) in 60 mL deionized or distilled water, and bring to a volume of 100 mL with sterile water. Store at 4 &#176;C (for a few months) or at -20 &#176;C (for years)</li>
	<li>Prepare 200 mL of 2% agarose gel. Use it fresh or, alternatively, store solidified at RT for up to several weeks.
	<ol>
		<li>Dissolve 4 g of agarose in 200 mL of 1x TBE buffer in a 600 mL beaker. Stir using a magnetic mixer for about 5 min until the agarose is completely suspended.</li>
		<li>Heat the 2% agarose solution in boiling-water or on a hot plate (for about 10 min, until the agarose is completely dissolved). Note that the beaker with the agarose gel must be covered with aluminum foil. Alternatively, heat the uncovered beaker in a microwave at a high temperature for about 3&#8211;5 min.</li>
		<li>Swirl the 2% agarose solution using a magnetic mixer, checking that the agarose is completely dissolved. 
		NOTE: Particles of agarose appear as translucent grains prior to complete dissolution. It may be necessary to reheat particles for several minutes (about 5&#8211;10 min).</li>
		<li>If a stored portion of the agarose gel is used, heat the beaker, covered with aluminum foil, in a hot-water bath (at about 60 &#176;C) until the agarose is dissolved. Remove with a Pasteur pipette any &#34;trace&#34; of solidified agarose from the surface prior to pouring.</li>
	</ol>
	</li>
</ol>
2. DNA Purification
<ol>
	<li>Perform the following before starting the purification:
	<ol>
		<li>Use human fresh whole blood samples, or thaw whole blood frozen samples quickly (for about 2&#8211;3 min) in a water bath (at 37 &#176;C) applying a mild agitation and then equilibrate to RT before use.</li>
		<li>Mix fresh or thawed blood samples inverting the tubes several times.</li>
	</ol>
	</li>
	<li>Start the purification procedure by following the supplier&#39;s protocols for 100 &#181;L of elution volume.</li>
	<li>Quantify and calculate the purity of DNA measuring the absorbance at 260, 280, and 320 nm.
	<ol>
		<li>Use sterile water to dilute the samples and to calibrate the spectrophotometer.</li>
		<li>Apply the following formula, in order to calculate the concentration of DNA sample = 50 &#181;g/mL x (A260 &#8722; A320) x dilution factor, and the purity of DNA = (A260 &#8722; A320)/(A280 &#8722; A320), with an acceptable ratio between 1.7 and 1.9.</li>
	</ol>
	</li>
</ol>
3. PCR Amplification of DNA Samples
<ol>
	<li>Prepare 25 &#181;L of reaction mixture in a 0.2 mL micro-amplification tube, as shown in Table 1.</li>
	<li>Carry out a PCR-amplification of the purified DNA samples using an automated thermal cycler, following the amplification program shown in Table 2.</li>
	<li>At the end of the PCR amplification, stop the PCR reactions by leaving the DNA samples at 4 &#176;C.</li>
</ol>
4. RFLP of PCR Products
<ol>
	<li>Prepare 22.5 &#181;L of master-mix solution for each sample, so that the selected restriction enzyme digests the PCR products, as shown in Table 3.</li>
	<li>Transfer 2.5 &#181;L of PCR product to a new PCR tube for each sample, using a pipette and filter tips.</li>
	<li>Add 22.5 &#181;L of digestion master-mix solution to the tubes containing the PCR product of each sample, using a pipette and filter tips.</li>
	<li>Incubate the reaction mixture (master-mix solution and PCR product) at 37 &#176;C for 4 h.</li>
</ol>
5. Gel Electrophoresis Analysis of PCR-RFLP Samples
<ol>
	<li>Stain the agarose gel by adding ethidium bromide (EtBr) at 0.5 &#181;g/mL to the gel for 10 min. EtBr binds to the DNA, which can be visualized under ultraviolet (UV) light. 
	Caution: It is important to use gloves and other protective devices during handling, storage, and disposal of EtBr, because it is a mutagen with potential carcinogenicity.</li>
	<li>Pour the prepared agarose gel into a gel tray with the well comb in place, and wait until it is solidified.</li>
	<li>Place the solidified agarose gel into the gel box (electrophoresis unit).</li>
	<li>Fill the gel box with 1x TBE until the gel is covered.</li>
	<li>With a pipette and filter tips, load 6 &#181;L of the amplified digest DNA (specifically, load 5 &#181;L of each sample and 1 &#181;L of gel loading dye) into the wells of the agarose gel. Add a DNA size marker in a separate well and in parallel with the samples.</li>
	<li>Run the gel for 20&#8211;30 min at 100 V.</li>
	<li>With a UV-transilluminator, visualize the cleaved DNA fragments or the undigested PCR products, comparing fragments with the DNA size marker, and register the results by photography following the manufacturer&#39;s instructions. 
	Caution: Use protective devices (safety glasses or a face mask) around UV light sources.</li>
</ol>

<ol>
	<li>Shen, R. F., Tai, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thromboxanes:+synthase+and+receptors.">Thromboxanes: synthase and receptors.</a> J Biomed Sci. 5 (3), 153-172 (1998).</li><li>Nusing, R. M., Hirata, M., Kakizuka, A., Eki, T., Ozawa, K., Narumiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+chromosomal+mapping+of+the+human+thromboxane+A2+receptor+gene.">Characterization and chromosomal mapping of the human thromboxane A2 receptor gene.</a> J Biol Chem. 268 (33), 25253-25259 (1993).</li><li>Cyrus, T., Ding, T., Pratic&#242;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+thromboxane+synthase,+prostacyclin+synthase+and+thromboxane+receptor+in+atherosclerotic+lesions:+correlation+with+plaque+composition.">Expression of thromboxane synthase, prostacyclin synthase and thromboxane receptor in atherosclerotic lesions: correlation with plaque composition.</a> Atherosclerosis. 208 (2), 376-381 (2010).</li><li>Cipollone, F., 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+Polymorphism+in+the+Cyclooxygenase+2+Gene+as+an+Inherited+Protective+Factor+Against+Myocardial+Infarction+and+Stroke.">A Polymorphism in the Cyclooxygenase 2 Gene as an Inherited Protective Factor Against Myocardial Infarction and Stroke.</a> JAMA. 291 (18), 2221-2228 (2004).</li><li>Fontana, 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=Identification+of+functional+polymorphisms+of+the+thromboxane+A2+receptor+gene+in+healthy+volunteers.">Identification of functional polymorphisms of the thromboxane A2 receptor gene in healthy volunteers.</a> Thromb Haemost. 96 (3), 356-360 (2006).</li><li>De Iuliis, V., 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=Differential+TBXA2+receptor+transcript+stability+is+dependent+on+the+C924T+polymorphism.+Prostaglandins+Other+Lipid+Mediat.">Differential TBXA2 receptor transcript stability is dependent on the C924T polymorphism. Prostaglandins Other Lipid Mediat.</a> pii. (17), (2017).</li><li>Saiki, R. 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=Enzymatic+amplification+of+beta-globin+genomic+sequences+and+restriction+site+analysis+for+diagnosis+of+sickle+cell+anemia.">Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.</a> Science. 230 (4732), 1350-1354 (1985).</li><li>Collins, F. S., Brooks, L. D., Chakravarti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+polymorphism+discovery+resource+for+research+on+human+genetic+variation.">A DNA polymorphism discovery resource for research on human genetic variation.</a> Genome Res. 8 (12), 1229-1231 (1998).</li><li>Lee, J. C. -. I., Chang, J. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABO+genotyping+by+polymerase+chain+reaction.">ABO genotyping by polymerase chain reaction.</a> J. Forensic Sci. 37 (5), 1269-1275 (1992).</li><li>Masao, O., Hirofumi, F., Jerzy, K. K., Hidetoshi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+detection+by+polymerase+chain+reaction-restriction+fragment+length+polymorphism.">Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism.</a> Nature Protocols. 2 (11), 2857-2864 (2007).</li><li>Iwasaki, H., 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=Accuracy+of+genotyping+for+single+nucleotide+polymorphisms+by+a+microarray-based+single+nucleotide+polymorphism+typing+method+involving+hybridization+of+short+allele-specific+oligonucleotides.">Accuracy of genotyping for single nucleotide polymorphisms by a microarray-based single nucleotide polymorphism typing method involving hybridization of short allele-specific oligonucleotides.</a> DNA Res. 9 (2), 59-62 (2002).</li><li>Papp, A. C., Pinsonneault, J. K., Cooke, G., Sadee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+allele-specific+PCR+and+fluorescence+melting+curves.">Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves.</a> Biotechniques. 34 (5), 1068-1072 (2003).</li><li>O'Meara, D., Ahmadian, A., Odeberg, J., Lundeberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+typing+by+apyrase-mediated+allele-specific+primer+extension+on+DNA+microarrays.">SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays.</a> Nucleic Acids Res. 30 (15), e75 (2002).</li><li>Pickering, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+DNA+ligation+and+rolling+circle+amplification+for+the+homogeneous,+end-point+detection+of+single+nucleotide+polymorphisms.">Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms.</a> Nucleic Acids Res. 30 (12), e60 (2002).</li><li>Chatterjee, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+sequencing+of+bacterial+and+P1+artificial+chromosome-nested+deletions+for+identifying+position-specific+single-nucleotide+polymorphisms.">Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.</a> Proc. Natl. Acad. Sci. USA. 96 (23), 13276-13281 (1999).</li><li>Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+discrimination+using+fluorogenic+probes+and+the+5'+nuclease+assay.">Allelic discrimination using fluorogenic probes and the 5' nuclease assay.</a> Genet. Anal. 14 (5-6), 143-149 (1999).</li><li>Haff, L. A., Smirnov, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleotide+polymorphism+identification+assays+using+a+thermostable+DNA+polymerase+and+delayed+extraction+MALDI-TOF+mass+spectrometry.">Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.</a> Genome Res. 7 (4), 378-388 (1997).</li><li>Gunderson, K. L., Steemers, F. L., Lee, G., Mendoza, L. G., Chee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+scalable+SNP+genotyping+assay+using+microarray+technology.">A genome-wide scalable SNP genotyping assay using microarray technology.</a> Nat. Genet. 37 (5), 549-554 (2005).</li></ol>

The authors have nothing to disclose.

In the present study, we have described a methodology that allows patient genotyping in order to investigate the post-transcriptional role of C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2R gene. First, this method relies on DNA extraction from whole blood. In particular, this first process consists of a purification of total human DNA, genomic and mitochondrial, from whole blood samples fresh or frozen, treated with EDTA (either citrate or heparin). For short term storage of whole blood samples, store at 2-8 &#176;C for up to 10 days. For storage over 10 days, store samples at -70 &#176;C. The automated purification process comprises 4 steps: lyse, bind, wash, and elute. Second, the method relies on PCR amplification of the TBXA2R gene portion containing the C924T mutation. Finally, identification of the wild type and/or mutant genotype using a restriction enzyme analysis (RFLP) on agarose gel is performed.
Critical steps in the protocol are the following: (i) In the case that a portion of the agarose gel stored at RT is used, the solidified agarose can be re-dissolved over a boiling-water bath (at 60 &#176;C for about 15-20 min) or in a microwave oven (3-5 min) prior to pouring. Note: loosen the cap when re-melting agarose in a bottle. (ii), furthermore, when re-heating agarose, evaporation will cause an increase of its concentration. For this reason, it could be useful to compensate by adding a small volume of water. (iii) DNA fragments of less than 1,000 bp were differentiated by agarose gel, and TBE buffer is recommended to obtain the best possible separation. (iv) We prefer to use an agarose gel rather than a polyacrylamide gel because the preparation of the latter is more difficult, and it takes much longer to set up. (v) The choice of the running time of the gel electrophoresis relies on the expected size of the amplification products. Based on this protocol, it is sufficient to perform an electrophoresis for 20-30 min at 100 V in 2% agarose gel, since the size of the PCR fragments ranges from 100-500 bp. (vi) It is mandatory to obtain 10 to 50 ng of a good quality template DNA extracted from human samples. For this reason, we prefer to use an automated DNA purification rather than a semi-automated or manual one. (vii) Prepare the master-mix reaction for PCR amplification and the master-mix solution for PCR products digestion, adding 10% more (to account for loss of liquid during pipetting) to the volume calculated multiplied by the number of samples for the volume required for one DNA sample.
The most frequent pitfall of the method is the presence of extra amplification products due to an incorrect thermal cycler program, an incorrect amplification master-mix preparation, or a DNA template contamination. Furthermore, the absence of PCR products may be due to inactivated Taq polymerase or an incorrect thermal cycler run. In addition, the presence of unexpected fragments may be due to PCR product contamination or to an incomplete digestion from either an inactivated enzyme, too little amount of restriction enzyme volume, or a too short incubation time.
In the past years, the following methodologies have been utilized for SNP analysis using the PCR technique: hybridization of short allele-specific oligonucleotides11, allele-specific PCR12, primer extension on DNA microarrays13, oligonucleotide ligation assay14, Direct DNA sequencing for identifying position-specific single-nucleotide polymorphisms15, Taqman method16, extraction Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry17, and GeneChips18. These methods are not ideal because they are not simple to use and/or require expensive equipment. Conversely, the PCR-RFLP method described in this study is inexpensive, simple to use, convenient, has a high efficiency, and allows rapid identification of the C924T polymorphism. A limitation to the present method is that it can only be used for a small number of SNPs and for a few samples in a working session.
For future applications, this method can be used for predictive studies concerning plaque formation and atherosclerosis progression by analyzing the patient genotypes for the TBXA2R C924T polymorphism. Furthermore, this method could identify subjects more susceptible to atherothrombotic processes, in particular, high-risk patients treated with aspirin. Finally, this method could be applied to study other polymorphisms involved in personalized medicine for specific drugs (for example, anticoagulants and anticonvulsants) in order to understand the appropriate drug dosage and the individual pharmacological and clinical response for each patient before beginning therapy and to avoid adverse effects.

TBXA2R is a member of the G-protein coupled superfamily with seven-transmembrane regions, which are widely expressed and localized either on the cell membranes or on intracellular structures1,2. The TBXA2R signaling pathway is involved in advanced atherosclerotic processes3. Increased expression of the TBXA2 receptor was demonstrated during the atherogenesis progression, and clinical and experimental studies showed its relevant role in ischemia and myocardial infarction4. C924T, a single nucleotide polymorphism (SNP) of the TBXA2R gene, was recognized as a functional polymorphism in healthy volunteers and has been linked to clinical disorders5. Furthermore, our previous study6 demonstrated that the C924T polymorphism of the TBXA2R gene is involved in transcript stability; specifically, there is an increased instability of the mutant (TT) type transcript with respect to the wild type (CC). In addition, several stimuli such as adenosine diphosphate (ADP), epinephrine, and collagen at different concentrations induced a platelet aggregation less effective for the mutant type (TT). This is consistent with a reduced thrombus formation and hemostasis. Thus, the instability of the TBXA2R transcript and the associated reduction of platelet aggregation might be associated with a protective role for the TBXA2R TT genotype against atherothrombosis and its complications in high-risk aspirin-treated patients6.
Here, we describe a methodology for patient genotyping to investigate the post-transcriptional role of C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on the following steps: (1) DNA extraction from whole blood, (2) PCR amplification of the TBXA2R gene portion containing the C924T mutation, and (3) identification of wild type and/or mutant genotypes using restriction fragment length polymorphism (RFLP) on agarose gel. RFLP is a technique that exploits variations in homologous DNA sequences7. This application was used to detect DNA polymorphisms, especially SNPs, and to find and associate biological relevance in genetic variations8. The polymorphism analyzed for the first time using the RFLP-PCR in humans was the ABO blood9. The RFLP-PCR method allows the analysis of genetic mutations in homologous DNA sequences by evaluating the presence of fragments of different lengths after the DNA digestion using highly specific restriction endonucleases10.
In past years, the following methodologies have been utilized for SNP analysis using the PCR technique: hybridization of short allele-specific oligonucleotides11, allele-specific PCR12, primer extension on DNA microarrays13, oligonucleotide ligation assay14, Direct DNA sequencing for identifying position-specific single-nucleotide polymorphisms15, Taqman method16, extraction Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry17, and GeneChips18. These techniques are not simple to use and may require expensive equipment. Conversely, the PCR-RFLP method is inexpensive, simple to use, convenient, has a high efficiency, and allows rapid identification of the C924T polymorphism. In addition, we confirmed the results by sequencing the TBXA2R gene using the Sanger method15.
This approach allows a predictive study of plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. This method could identify subjects more susceptible to atherothrombotic processes, in particular those among high-risk, aspirin-treated patients.

<table><tbody><tr><td>QIAsymphony SP</td><td>QIAGEN</td><td>937055</td><td></td></tr><tr><td>Spectrophotometer</td><td>EPPENDORF</td><td>6131-02222</td><td></td></tr><tr><td>UV-transilluminator</td><td>UVP</td><td>732-110</td><td></td></tr><tr><td>PCR tubes</td><td>EPPENDORF</td><td>H0030121589</td><td></td></tr><tr><td>PCR thermal cycler</td><td>EPPENDORF</td><td>5331-03721</td><td></td></tr><tr><td>Pipettors and filter tips</td><td>EPPENDORF</td><td>H4910000018/42/69 AND 0030067037/10/02</td><td></td></tr><tr><td>Horizontal minigel electrophoresis apparatus</td><td>DIATECH PHORESIS 10</td><td>RI002-10</td><td></td></tr><tr><td>Dry block heater</td><td>TWIN INCUBATOR</td><td>DG210</td><td></td></tr><tr><td>QIAsymphony DNA Midi Kit</td><td>QIAGEN</td><td>931255</td><td></td></tr><tr><td>10x PCR buffer (usually supplied by the manufacturer with the Taq polymerase)</td><td>DIATECH AND TAKARA</td><td>T0100 AND R0001DM</td><td></td></tr><tr><td>Taq polymerase</td><td>TAKARA</td><td>R0001DM</td><td></td></tr><tr><td>dNTP mixture</td><td>DIATECH&amp;nbsp; pharmacogenetics</td><td>NM001</td><td>dNTP MIX 10x 100 microliters, 10 mM</td></tr><tr><td>PCR primers</td><td>DIATECH&amp;nbsp; pharmacogenetics</td><td>\\</td><td></td></tr><tr><td>Restriction enzyme RsaI</td><td>New England biolabs</td><td>R0167L</td><td></td></tr><tr><td>Restriction enzyme 10x buffer</td><td>New England biolabs</td><td>R0167L</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td>DNA fragments are best separated in TBE buffer</td></tr><tr><td>Tris base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B7901</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Sigma</td><td>E7889</td><td></td></tr><tr><td>10% (wt/vol) ammonium persulfate</td><td>Sigma</td><td>E3678</td><td>prepared fresh each time</td></tr><tr><td>EtBr (0.5 &amp;mu;g/&amp;mu;L)</td><td>Sigma</td><td>E8751</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>B0126</td><td></td></tr><tr><td>Xylene cyanol FF</td><td>Sigma</td><td>X4126</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>DNA size marker</td><td>DIATECH&amp;nbsp; pharmacogenetics</td><td>R1002-10</td><td>Plasmide pBluescript II SK (+) restrict MSPI</td></tr><tr><td>Sterile water (autoclaved)</td><td>DIATECH&amp;nbsp; pharmacogenetics</td><td>\\</td><td></td></tr></tbody></table>

a method to study the c924t polymorphism of the thromboxane a2 receptor gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

The goal of this method is to evaluate the Thromboxane A2 receptor genotype with regard to the C924T polymorphism. The human TBXA2R gene is located on 19p13.3, spans 15 kbp, and consists of three exons separated by two introns. The C924T polymorphisms of the TBXA2R gene (of 539 bp) was amplified using the PCR primers shown in Figure 1, which were well-engineered to amplify a specific DNA region but not an orthologous or paralogous nonspecific region. In addition, an RFLP analysis using an RsaI restriction enzyme (Figure 1) on the PCR products was performed and the results were visualized on an agarose gel, in order to characterize the specific studied SNP.
Based on the presence of different fragment lengths after digestion of the DNA, it is possible to discriminate a patient&#39;s genotype for the C924T polymorphism. In fact, as shown on Figure 2, the homozygosity of the major allele (CC) displays two bands (395 and 144 bp), because the restriction enzyme precisely cuts the TBXA2R gene portion at the C924T polymorphism site. The homozygosity of the minor allele (TT) is demonstrated by a single band (539 bp), because the restriction enzyme cutting does not occur. The heterozygous (CT) allele shows three bands (539, 395, and 144 bp). As shown in Figure 2, the C924T polymorphism, defined by RsaI digestion on PCR product, has been confirmed by sequence analysis.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>10x PCR buffer Tween-20 (15 M MgCl2)</td>
			<td>0.25</td>
			<td>1.5 MgCl2 mmol/L</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM)</td>
			<td>1</td>
			<td>200 &#181;M</td>
		</tr>
		<tr>
			<td>Primer (forward)(10 pmol/&#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M/&#181;L</td>
		</tr>
		<tr>
			<td>Primer (reverse)(10 pmol/&#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M/&#181;L</td>
		</tr>
		<tr>
			<td>Taq polymerase (5 U/&#181;L)</td>
			<td>0.2</td>
			<td>1 U/&#181;L</td>
		</tr>
		<tr>
			<td>Sample DNA (42 ng/&#181;L)</td>
			<td>1</td>
			<td>42 ng/&#181;L</td>
		</tr>
		<tr>
			<td>DNase-free-water</td>
			<td>20.55</td>
			<td></td>
		</tr>
		<tr>
			<td>Total</td>
			<td>25</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: PCR amplification setup. A 25 &#181;L master-mix reaction mixture set up in a 0.2 mL PCR tube to amplify a single DNA sample.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Standard PCR</td>
		</tr>
		<tr>
			<td>Initial activating step</td>
			<td>5 min</td>
			<td></td>
			<td>95 &#176;C</td>
		</tr>
		<tr>
			<td>3-step cycling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Denaturation</td>
			<td>30 s</td>
			<td></td>
			<td>94 &#176;C</td>
		</tr>
		<tr>
			<td>Annealing</td>
			<td>60 s</td>
			<td></td>
			<td>55 &#176;C</td>
		</tr>
		<tr>
			<td>Extension</td>
			<td>60 s</td>
			<td></td>
			<td>72 &#176;C</td>
		</tr>
		<tr>
			<td>Number of cycles</td>
			<td></td>
			<td>30 cycles</td>
			<td></td>
		</tr>
		<tr>
			<td>Final extension</td>
			<td>8 min</td>
			<td></td>
			<td>72 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 2: PCR amplification program. Set up an automated thermal cycler in order to perform PCR and amplify the template DNA. PCR reactions are stopped by chilling at 4 &#176;C.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L) (n = 1)</td>
			<td>Volume (&#181;L) (n = 10)*</td>
		</tr>
		<tr>
			<td>10x enzyme buffer</td>
			<td>2.5</td>
			<td>27.5</td>
		</tr>
		<tr>
			<td>Restriction enzyme (5 U/&#181;L)</td>
			<td>0.5</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>DNase-free-water</td>
			<td>19.5</td>
			<td>214.5</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>22.5</td>
			<td>247.5</td>
		</tr>
	</tbody>
</table>
Table 3: Restriction enzyme digestion of PCR products. A master-mix solution is prepared so that the selected restriction enzyme digests the PCR products. *: To set up a master-mix solution for 10 samples, add 10% more, finally making up 11 samples.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57289/57289fig1v2.jpg" /> 
Figure 1: PCR primers and RsaI restriction enzyme. The forward and reverse primers designed for amplifying the TBXA2R gene portion of 539 bp containing the C924T polymorphism. RsaI is the restriction enzyme selected to recognize the GTAC sites. &#709;: cutting site of RsaI enzyme. C(/T): C924T polymorphism. <a href="https://cloudflare.jove.com/files/ftp_upload/57289/57289fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57289/57289fig2v2.jpg" /> 
Figure 2: Electrophoretic pattern and DNA sequence analysis. (A) The C924T genotype is recognized by the RFLP pattern after digestion with the specific RsaI enzyme. (B) DNA sequence analysis: The results obtained by RFLP after digestion with the RsaI restriction enzyme were confirmed using sequence analysis showing the C924T polymorphism. This figure has been modified from De Iuliis et al., Prostaglandins &#38; Other Lipid Mediators6. <a href="https://cloudflare.jove.com/files/ftp_upload/57289/57289fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene at JoVE.com

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

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Research

JoVE Journal

Genetics

10.0K Views.  University of Chieti. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het trombose- en hemostaseveld met kenmerken die gevoeliger zijn voor atherothrombotische processen, met name patiënten met een hoog risico die met aspirine worden behandeld. Het belangrijkste voordeel van deze techniek is dat het goedkoop, eenvoudig te gebruiken, handig, heeft een hoge efficiëntie en maakt een snelle identificatie van de C924T polymorfismen. Hoewel deze methode inzicht kan geven in het atherothromboseve...

Video: A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Biology

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

Immunology and Infection

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Neuroscience

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin amyloidosis (ATTR). ATTR is a rare but fatal disease with heterogeneous phenotypes; therefore, the diagnosis is sometimes delayed. With increasing attention and broader recognition on early manifestations of ATTR as well as emerging treatments, appropriate diagnostic studies, including the transthyretin (TTR) genetic test, to confirm the types and variants of ATTR are therefore fundamental to improve the prognosis. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutations much more quickly and safer than conventional methods such as southern blot. Herein, we demonstrate genetic confirmation of the ATTR Ala97Ser mutation, the most common endemic mutation in Taiwan. The protocol comprises four main steps: collecting whole blood specimen, DNA extraction, genetic analysis of all four TTR exons with PCR, and DNA sequencing.

Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

Medicine

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

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