Name: high-resolution melting pcr for complement receptor 1 length polymorphism genotyping: an innovative tool for alzheimer's disease gene susceptibility assessment
Uploaded: 2017-07-18T14:00:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessmen

我们感谢Immunologie Innovante的所有成员，免疫学系的工作人员以及内科和老年医学系的工作人员，他们为协议的优化和验证作出了贡献。这项工作由兰斯大学医院资助（拨款号为AOL11UF9156）。我们还感谢Fiona Ecarnot（EA3920，法国Besancon大学医院）的社论援助。

人类血液采集和处理方案由区域伦理委员会（CPP Est II）审议通过，方案编号为2011年A00594-37。 注意:以下协议描述了人体血液的处理。处理生物危害材料时，请遵循制度准则。应穿戴实验室外套和手套等实验室安全设备。描述协议的流程图如图3所示 。 血液和体液DNA提取<ol><li>将20μL蛋白酶K（15μg/μL）吸入1.5 mL管的底部。 </li><li>加入200μL样品至1.5 mL管中。在200μL的PBS中使用多达200μL的全血，血浆，血清，血沉棕黄层或体液，或最多5×10 6个淋巴细胞。 </li><li>向样品中加入200μL裂解缓冲液。通过脉冲涡旋混合15秒。 </li><li>在水浴中在56℃下孵育10分钟。 </li><li>简单地离心1.5 mL管，以从盖子的内部除去液滴。 </li><li>向样品中加入200μL乙醇（96-100％），并通过脉冲涡旋再次混合15秒。混合后，短暂离心1.5 mL管，以从盖子的内部除去液滴。 </li><li>小心地将来自步骤1.6的混合物施加到膜柱（在2mL收集管中）。不润湿轮辋，关闭盖子并以6,000 xg离心1分钟。将膜柱置于干净的2-mL收集管中，并丢弃含有滤液的管。 </li><li>仔细打开膜柱，加入500μL洗涤缓冲液1，不会弄脏边缘。关闭盖子并以6,000 xg离心1分钟。将膜柱放入干净的2 mL收集管中，弃去含有滤液的试管。 </li><li>仔细打开膜柱，加入500μL洗涤缓冲液2，不加湿e边缘关闭盖子并全速离心（20,000 xg）3分钟。 </li><li>将膜柱置于新的2 mL收集管中，并用滤液弃去收集管。以20,000 xg离心1分钟。 </li><li>将膜柱放入干净的1.5 mL管中，弃去含有滤液的收集管。 </li><li>小心打开膜柱，加入200μL蒸馏水。在室温下孵育5分钟，然后以6000×g离心1分钟。 </li><li>继续测定DNA浓度步骤或在-20℃冷冻DNA样品</li></ol> 2. DNA浓度的测定注意: 见图4 。 <ol><li>按1在分光光度计上选择DNA模式。使用左右箭头选择5 mm路径长度。使用左右箭头选择1的稀释因子。 </li><li>使用th选择单位（μg/ mL）e左右箭头。使用左右箭头选择50的因子。按OK 。 </li><li>将10μL蒸馏水吸入比色皿。将比色杯放入分光光度计。按OA / 100％T按钮。 </li><li>移取10μLDNA样品（1/4稀释于蒸馏水中）至比色杯中。将比色杯放入分光光度计。按绿色按钮（读/开）。注意浓度。 </li><li>在-20°C冷冻DNA样品。 </li></ol> 3. HRM-PCR协议<ol><li>解冻DNA样品。用水稀释1.5 mL管中的DNA样品，调整浓度为10 ng /μL 注意:稀释的DNA的总体积应在2μL和10μL之间。 </li><li>解冻底漆溶液。用水稀释1.5 mL管中的底物溶液，使其浓度达到6μM。 注意:引物序列和反应条件在T中提供能够1。 </li></ol><img alt="表格1" src="/files/ftp_upload/56012/56012table1.jpg" /> 表1:高分辨率熔解分析中使用的引物和参数。 <ol start="3"><li>解冻HRM-PCR试剂盒解决方案，并通过涡旋仔细混合，以确保所有内容物的恢复。在打开它们之前，将微量离心机中的含有酶结合染料，MgCl 2和水的三个小瓶简单旋转。将它们储存在室温下。 </li><li> 在室温的1.5 mL管中，通过以下列顺序添加以下组分，制备PCR混合物，进行一次20μL的反应: <ol><li> 10μL与DNA结合染料的酶混合物; </li><li> 2μL25mM MgCl 2 ; </li><li> 1μL引物1,6μM（终浓度:300nM）; </li><li> 1μL引物2，6μM（终浓度:300纳米）;和</li><li> 5μL水。 注意:为了制备多于一种反应的PCR混合物，将上述体积乘以待运行的反应次数加上另外一个反应。 </li></ol></li><li>通过涡旋仔细混合。 </li><li>吸管将19μL上文制备的PCR混合物加入到白色多孔板的每个孔中。 </li><li>加入1μL浓度调节DNA模板，步骤3.1中制备。 注意:对于控制反应，始终使用样品运行阴性对照。为了制备阴性对照，用水替代模板DNA。 </li><li>用密封箔密封白色多孔板。 </li><li>将白色多孔板放入离心机中，并将其与适当的配重（ 即另一个多孔板）平衡。在含有适合适配器的多孔板的转子的标准摆式离心机中，以1500xg离心1分钟。 </li><li>将白色多孔板装入HRM-PCR仪器。</li><li>启动具有以下PCR条件的HRM-PCR程序: 变性:95℃10分钟; 1个周期。 扩增:95℃10秒，62℃15秒，72℃20秒; 47个周期。 熔点:95℃;斜坡率:0.02°C / s;每摄氏25次; 1个周期。 冷却:40℃30秒;斜坡率为2.2°C / s; 1个周期。 </li></ol> 4.人力资源管理分析确定CR1长度多态性注意:尽管可以使用其他软件包，但描述的方法（ 图5 ）是我们软件的特定（见材料表 ）。 <ol><li>打开基因扫描软件进行CR1长度多态性扫描分析。 </li><li>打开包含扩增程序和熔解曲线程序的实验。 </li><li>单击模块栏中的样品编辑器 ，然后选择S罐头工作流程。 </li><li>定义样本的属性（ 即名称，未知或阴性对照）。 </li><li>单击模块栏中的分析 。 </li><li>在创建新分析列表中，选择基因扫描。 </li><li>单击" 规范化"选项卡以对熔解曲线进行归一化。 </li><li>单击温度移位选项卡以重置熔解曲线的温度轴（x轴）。 注意:下图显示了正常化和温度偏移的熔融曲线。 </li><li>单击" 计算"按钮分析结果并确定分组。 </li><li>单击图表区域中的差异绘图选项卡以查看归一化和移位熔融曲线以及归一化和温度移位差分图 。 </li></ol>

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	<li>Prince, 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=World+Alzheimer+Report+2015,+The+Global+Impact+of+Dementia:+An+analysis+of+prevalence,+incidence,+cost+and+trends.">World Alzheimer Report 2015, The Global Impact of Dementia: An analysis of prevalence, incidence, cost and trends.</a> Alzheimer's Disease International. , (2015).</li><li>McKhann, G. 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=The+diagnosis+of+dementia+due+to+Alzheimer's+disease:+recommendations+from+the+National+Institute+on+Aging-Alzheimer's+Association+workgroups+on+diagnostic+guidelines+for+Alzheimer's+disease.">The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.</a> Alzheimers Dement. 7 (3), 263-269 (2011).</li><li>Serrano-Pozo, A., Frosch, M. P., Masliah, E., Hyman, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+alterations+in+Alzheimer+disease.">Neuropathological alterations in Alzheimer disease.</a> Cold Spring Harb Perspect Med. 1 (1), 006189 (2011).</li><li>Goate, A., 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=Segregation+of+a+missense+mutation+in+the+amyloid+precursor+protein+gene+with+familial+Alzheimer's+disease.">Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.</a> Nature. 349 (6311), 704-706 (1991).</li><li>Sherrington, 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=Cloning+of+a+gene+bearing+missense+mutations+in+early-onset+familial+Alzheimer's+disease.">Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.</a> Nature. 375 (6534), 754-760 (1995).</li><li>Levy-Lahad, E., 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+familial+Alzheimer's+disease+locus+on+chromosome+1.">A familial Alzheimer's disease locus on chromosome 1.</a> Science. 269 (5226), 970-973 (1995).</li><li>Mayeux, R., Stern, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Alzheimer+disease.">Epidemiology of Alzheimer disease.</a> Cold Spring Harb Perspect Med. 2 (8), (2012).</li><li>Corder, E. 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=Gene+dose+of+apolipoprotein+E+type+4+allele+and+the+risk+of+Alzheimer's+disease+in+late+onset+families.">Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families.</a> Science. 261 (5123), 921-923 (1993).</li><li>Yu, J. T., Tan, L., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+in+Alzheimer's+disease:+an+update.">Apolipoprotein E in Alzheimer's disease: an update.</a> Annu Rev Neurosci. 37, 79-100 (2014).</li><li>Lambert, J. C., 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=Genome-wide+association+study+identifies+variants+at+CLU+and+CR1+associated+with+Alzheimer's+disease.">Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease.</a> Nat Genet. 41 (10), 1094-1099 (2009).</li><li>Liu, D., Niu, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure,+genetic+polymorphisms,+expression+and+biological+functions+of+complement+receptor+type+1+(CR1/CD35).">The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35).</a> Immunopharmacol Immunotoxicol. 31 (4), 524-535 (2009).</li><li>Jacquet, 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=Deciphering+complement+receptor+type+1+interactions+with+recognition+proteins+of+the+lectin+complement+pathway.">Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.</a> J Immunol. 190 (7), 3721-3731 (2013).</li><li>Pham, B. N., 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=Analysis+of+complement+receptor+type+1+expression+on+red+blood+cells+in+negative+phenotypes+of+the+Knops+blood+group+system,+according+to+CR1+gene+allotype+polymorphisms.">Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms.</a> Transfusion. 50 (7), 1435-1443 (2010).</li><li>Cosio, F. G., Shen, X. P., Birmingham, D. J., Van Aman, M., Hebert, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+mechanisms+responsible+for+the+reduction+in+erythrocyte+complement+receptors+when+immune+complexes+form+in+vivo+in+primates.">Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates.</a> J Immunol. 145 (12), 4198-4206 (1990).</li><li>Krych-Goldberg, M., Moulds, J. M., Atkinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+complement+receptor+type+1+(CR1)+binds+to+a+major+malarial+adhesin.">Human complement receptor type 1 (CR1) binds to a major malarial adhesin.</a> Trends Mol Med. 8 (11), 531-537 (2002).</li><li>Cohen, J. H., Geffriaud, C., Caudwell, V., Kazatchkine, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+CR1+(the+C3b+complement+receptor,+CD35)+expression+on+erythrocytes+of+HIV-infected+individuals.">Genetic analysis of CR1 (the C3b complement receptor, CD35) expression on erythrocytes of HIV-infected individuals.</a> Aids. 3 (6), 397-399 (1989).</li><li>Rogers, J., 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=Complement+activation+by+beta-amyloid+in+Alzheimer+disease.">Complement activation by beta-amyloid in Alzheimer disease.</a> Proc Natl Acad Sci USA. 89 (21), 10016-10020 (1992).</li><li>Rogers, J., 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=Peripheral+clearance+of+amyloid+beta+peptide+by+complement+C3-dependent+adherence+to+erythrocytes.">Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes.</a> Neurobiol Aging. 27 (12), 1733-1739 (2006).</li><li>Krych-Goldberg, M., Atkinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+complement+receptor+type+1.">Structure-function relationships of complement receptor type 1.</a> Immunol Rev. 180, 112-122 (2001).</li><li>Klickstein, L. B., 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=Human+C3b/C4b+receptor+(CR1).+Demonstration+of+long+homologous+repeating+domains+that+are+composed+of+the+short+consensus+repeats+characteristics+of+C3/C4+binding+proteins.">Human C3b/C4b receptor (CR1). Demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristics of C3/C4 binding proteins.</a> J Exp Med. 165 (4), 1095-1112 (1987).</li><li>Hourcade, D., Miesner, D. R., Atkinson, J. P., Holers, V. M. <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+an+alternative+polyadenylation+site+in+the+human+C3b/C4b+receptor+(complement+receptor+type+1)+transcriptional+unit+and+prediction+of+a+secreted+form+of+complement+receptor+type+1.">Identification of an alternative polyadenylation site in the human C3b/C4b receptor (complement receptor type 1) transcriptional unit and prediction of a secreted form of complement receptor type 1.</a> J Exp Med. 168 (4), 1255-1270 (1988).</li><li>Krych, M., Hourcade, D., Atkinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sites+within+the+complement+C3b/C4b+receptor+important+for+the+specificity+of+ligand+binding.">Sites within the complement C3b/C4b receptor important for the specificity of ligand binding.</a> Proc Natl Acad Sci USA. 88 (10), 4353-4357 (1991).</li><li>Krych-Goldberg, 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=Decay+accelerating+activity+of+complement+receptor+type+1+(CD35).+Two+active+sites+are+required+for+dissociating+C5+convertases.">Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases.</a> J Biol Chem. 274 (44), 31160-31168 (1999).</li><li>Klickstein, L. B., Barbashov, S. F., Liu, T., Jack, R. M., Nicholson-Weller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+receptor+type+1+(CR1,+CD35)+is+a+receptor+for+C1q.">Complement receptor type 1 (CR1, CD35) is a receptor for C1q.</a> Immunity. 7 (3), 345-355 (1997).</li><li>Ghiran, I., 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=Complement+receptor+1/CD35+is+a+receptor+for+mannan-binding+lectin.">Complement receptor 1/CD35 is a receptor for mannan-binding lectin.</a> J Exp Med. 192 (12), 1797-1808 (2000).</li><li>Cornillet, P., Philbert, F., Kazatchkine, M. D., Cohen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+determination+of+the+CR1+(CD35)+density+polymorphism+on+erythrocytes+using+polymerase+chain+reaction+amplification+and+HindIII+restriction+enzyme+digestion.">Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion.</a> J Immunol Methods. 136 (2), 193-197 (1991).</li><li>Moulds, J. M., Nickells, M. W., Moulds, J. J., Brown, M. C., Atkinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C3b/C4b+receptor+is+recognized+by+the+Knops,+McCoy,+Swain-langley,+and+York+blood+group+antisera.">The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera.</a> J Exp Med. 173 (5), 1159-1163 (1991).</li><li>Moulds, J. M., Moulds, J. J., Brown, M., Atkinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiglobulin+testing+for+CR1-related+(Knops/McCoy/Swain-Langley/York)+blood+group+antigens:+negative+and+weak+reactions+are+caused+by+variable+expression+of+CR1.">Antiglobulin testing for CR1-related (Knops/McCoy/Swain-Langley/York) blood group antigens: negative and weak reactions are caused by variable expression of CR1.</a> Vox Sang. 62 (4), 230-235 (1992).</li><li>Wilson, J. 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=Identification+of+a+restriction+fragment+length+polymorphism+by+a+CR1+cDNA+that+correlates+with+the+number+of+CR1+on+erythrocytes.">Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes.</a> J Exp Med. 164 (1), 50-59 (1986).</li><li>Wong, W. W., 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=Structure+of+the+human+CR1+gene.+Molecular+basis+of+the+structural+and+quantitative+polymorphisms+and+identification+of+a+new+CR1-like+allele.">Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identification of a new CR1-like allele.</a> J Exp Med. 169 (3), 847-863 (1989).</li><li>Herrera, A. H., Xiang, L., Martin, S. G., Lewis, J., Wilson, 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=Analysis+of+complement+receptor+type+1+(CR1)+expression+on+erythrocytes+and+of+CR1+allelic+markers+in+Caucasian+and+African+American+populations.">Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in Caucasian and African American populations.</a> Clin Immunol Immunopathol. 87 (2), 176-183 (1998).</li><li>Rowe, J. A., 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=Erythrocyte+CR1+expression+level+does+not+correlate+with+a+HindIII+restriction+fragment+length+polymorphism+in+Africans;+implications+for+studies+on+malaria+susceptibility.">Erythrocyte CR1 expression level does not correlate with a HindIII restriction fragment length polymorphism in Africans; implications for studies on malaria susceptibility.</a> Genes Immun. 3 (8), 497-500 (2002).</li><li>Birmingham, D. J., 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+type+one+complement+receptor+(CR1)+involves+an+additional+cysteine+within+the+C3b/C4b+binding+domain+that+inhibits+ligand+binding.">A polymorphism in the type one complement receptor (CR1) involves an additional cysteine within the C3b/C4b binding domain that inhibits ligand binding.</a> Mol Immunol. 44 (14), 3510-3516 (2007).</li><li>Venter, J. C., 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=The+sequence+of+the+human+genome.">The sequence of the human genome.</a> Science. 291 (5507), 1304-1351 (2001).</li><li>Dwight, Z., Palais, R., Wittwer, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=uMELT:+prediction+of+high-resolution+melting+curves+and+dynamic+melting+profiles+of+PCR+products+in+a+rich+web+application.">uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application.</a> Bioinformatics. 27 (7), 1019-1020 (2011).</li><li>Brouwers, N., 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=Alzheimer+risk+associated+with+a+copy+number+variation+in+the+complement+receptor+1+increasing+C3b/C4b+binding+sites.">Alzheimer risk associated with a copy number variation in the complement receptor 1 increasing C3b/C4b binding sites.</a> Mol Psychiatry. 17 (2), 223-233 (2012).</li><li>Hazrati, L. N., 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=Genetic+association+of+CR1+with+Alzheimer's+disease:+a+tentative+disease+mechanism.">Genetic association of CR1 with Alzheimer's disease: a tentative disease mechanism.</a> Neurobiol Aging. 33 (12), 2945-2949 (2012).</li><li>Mahmoudi, 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=Alzheimer's+disease+is+associated+with+low+density+of+the+long+CR1+isoform.">Alzheimer's disease is associated with low density of the long CR1 isoform.</a> Neurobiol. Aging. 36, 1712-1765 (2015).</li><li>Helgeson, M., Swanson, J., Polesky, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knops-Helgeson+(Kna),+a+high-frequency+erythrocyte+antigen.">Knops-Helgeson (Kna), a high-frequency erythrocyte antigen.</a> Transfusion. 10 (3), 137-138 (1970).</li></ol>

作者是兰斯香槟 - 阿登大学（URCA）拥有的专利的发明人（专利号:WO 2015166194）

在这里，我们描述了一种广泛使用的研究CR1长度多态性的方法。用于扩增或分段杂交的分子生物学技术从未能够给出令人满意的结果，其允许确定所有个体的CR1长度多态性。这是因为CR1基因的重复结构（ 即 CR1的高度重复的SCR）。这里描述的分子生物学方法利用了CR1分子中LHR-B的定量分布。 CR1分子包括n个LHR-B结构域，其中n是0和3之间的数字，以及仅有一个LHR-C结构域， 如图2所述和所示。定量域B检测使得可以完美地区分一个附加的或缺失的域B. 从提取的遗传物质中，使用单对特异性引物通过PCR扩增来自LHR-B的第一个DNA片段，表达将LHR-B的特征变体称为"变体扩增子B"。属于LHR-C的第二个DNA片段称为"不变扩增子"也被扩增。该第二DNA片段是LHR-C的片段，但也可以使用LHR-A或-D的另一种不变片段。 通过相同的引物扩增两个DNA片段，变体扩增子B和不变扩增子，并显示核苷酸序列的五个差异。该特征使得在后续步骤中可以使用定量分子生物学工具HRM来确定变体扩增子B的数量和不变扩增子的数目，HRM适用于此目的。扩增子B的数量与不变扩增子的比例（比例:扩增子B /不变扩增子）根据CR1分子的长度而变化。实际上，CR1 * 4显示3个扩增子B单位至1个不变扩增子，CR1 * 2显示2个扩增子B单位至1个不变扩增子，CR1 * 1显示1个扩增子B至1个不变扩增子，CR1 * 3显示0个扩增子B单位至1个不变扩增子（ 图2 ）。因此，该HRM-PCR方法能够进行CR1长度多态性的基因分型。 然而，在研究开始时，该技术需要使用参考对象，其CR1表型已知（ 即，由WB建立），以便能够根据获得的曲线的参考分布来建立CR1的基因分型通过HRM-PCR。可以使用两种类型的表示，即"归一化和转移熔融曲线"和"归一化和温差变化差异图"。它们根据WB获得的CR1长度多态性表型显示不同组的曲线轮廓（ 图6 ）。从"归一化和转移熔融曲线"步骤，我们的方法可以区别于此对应于CR1的同种型的不同组的曲线，按颜色分组。然而，对应于不同数学表示的"归一化和临时移位差异图"允许更容易地显示不同组的曲线。虽然在本研究中常规使用制造商的软件，但其他软件（如uANALYSE）也可用作数据提取程序进行曲线分析。 迄今为止，WB分析技术是能够鉴定蛋白质水平上不同CR1长度多态性的原始技术。然而，这种技术有许多缺点。特别是WB的蛋白质分析只能在提取细胞膜后进行。因此，它是复杂和非常耗时的。此外，该技术需要在程序开始时在适当的条件下获得新鲜的血液样品以实现有用的resULTS。相反，使用DNA进行HRM-PCR可以在长期储存后甚至使用干血斑或样品。 HMR-PCR需要专门的DNA融合仪器，专用仪器或具有HRM软件的HMR容量热循环仪。 SNP检测取决于几个因素:i）大PCR片段中包含的SNP比小PCR片段中相同的SNP更难检测; ii）属于III类和IV类的SNP比I和II类更难检测34 ;和iii）无论HMR平台的分辨力如何，不能通过熔化来排列侧接最近邻碱基对称（ 例如 5&#39;-G（G / C）C-3&#39;）的SNP。使用uMELT软件35测试含有感兴趣的SNP的预测的PCR片段以评估测定的有效性可能是有用的。最后，HMR-PCR是一种类比法，意味着熔解曲线的相似性下降参考文献和模板并不一定意味着模板序列与参考文献相同，因为模板序列可能与引用不同，但在热力学上是等同的。然而，这些限制不适用于这里描述的方法，其基于5个核苷酸序列不同的扩增子的混合物的融合。 另外，这里描述的技术基于人力资源管理分析，具有很多优点。首先，CR1长度多态性被更快地确定，因为一旦提取遗传物质已经在约2小时内获得结果。相反，基于WB蛋白质分析的传统生物化学技术需要相对长的步骤:通过丙烯酰胺凝胶电泳细胞膜提取物，将蛋白质转移到硝酸纤维素或聚偏二氟乙烯（PVDF）膜的步骤，以及鉴定异构体CR1分子通过免疫化学发光。第二，HRM-PCR技术还具有不太昂贵的优点，这对于可能应用于许多个体（ 即，大尺度）的方法尤其重要，以确定它们对诸如阿尔茨海默氏病等疾病的发展的易感性，狼疮或疟疾。 近来，我们和其他人已经表明，CR1 * 2同种型，其包含一个额外的C3b / C4b结合位点，用AD 36，37，38相关联。在我们以前发表的研究中，基因和蛋白质水平的CR1长度多态性在98.9％的受试者中是一致的38 。当通过HRM-PCR获得的长度多态性与通过WB获得的长度多态性比较时获得的不一致结果对应于具有通过HRM（基因）但是ex（CR1 * 1，CR1 * 2）基因型谱的受试者按照WB（蛋白质）仅在红细胞表面按下CR1 * 1同种型。在我们的研究中，当WB以更长的暴露时间进行时，缺乏CR1 * 2同种型表达（具有无声等位基因的个体）是可重现的，并且可以通过存在沉默的CR1等位基因（CR1 * 2）来解释，描述在Helgeson 39的文献中。然而，需要进一步研究较大的人口来支持这一假设。 应当注意，私人SNP（仅出现在小家族组或甚至单个个体中的SNP）导致不同的曲线轮廓，应该使用参考表型确定（WB）进行解密，但不能与规范轮廓混淆以避免任何对CR1长度多态性基因型的误解。

<html> AD，痴呆症的最常见原因，影响到世界各地的30多万人口，是一个重大的公共卫生问题1。临床上，AD的特征在于神经认知障碍导致自主进展性丧失2 。 AD具有两个神经病理学特征，即细胞外淀粉样蛋白沉积和细胞内神经原纤维缠结3 。 传统上，根据疾病发病年龄，AD被分为两种形式。首先是早发型AD（EOAD），其发病最常发生在65岁之前;这种形式占AD病例的5％以下。它是AD的罕见的常染色体显性形式，其导致在淀粉样蛋白前体蛋白（ APP） 4 ，早老素1（ PSEN1 ） 5或早老素2（ PSEN2 ）中的完全渗透突变，&gt; 6个基因。第二，更常见的疾病形式（&gt; 90％的AD病例）称为"偶发性"迟发性AD（LOAD），最常见于65岁或以上的个体。这是由于多种遗传和环境危险因素7。在LOAD，载脂蛋白E（APOE）基因的等位基因4是主要遗传危险因素8,9。此外，通过全基因组关联研究（GWAS）鉴定了20多个基因位点与AD的风险相关，其中一个位于位于上游的补体成分（3b / 4b）受体1（ CR1 ）基因10染色体1q32在补体相关蛋白质簇中。 CR1基因编码补体受体1型（CR1）蛋白，补体活性调节剂的一个组成部分。 CR1（C3b / C4b受体，CD35），近似的跨膜糖蛋白ely 200 kDa 11 ，结合C3b，C4b，C3bi，C1q，甘露聚糖结合凝集素（MBL）和ficolin补体蛋白12 。 CR1的生物学功能随着其表达的细胞类型而变化。在人类中，总循环CR1的90％存在于红细胞（RBC） 13中 。存在于红细胞表面，CR1与C3b-或C4b-调理微生物或免疫复合物结合，有助于其循环清除。势必CR1配合确实被转移到吞噬红细胞的时候都要经过肝脏和脾脏11,14。通过限制C3b和C4b的沉积，CR1可能会阻止过度的补体活化。因此，CR1在RBC上的表达被认为是保护组织，如脑神经系统，免疫复合物沉积和所产生的疾病的重要元素。 RBC上的CR1也是已知的病原感染15起着重要的作用，16。此外，作为先天免疫力的关键因素，CR1参与了补体级联的调控和免疫复合物的运输和清除。 CR1通过结合C3b和C4b片段并解离经典和替代转化酶（C2a与C4b2a复合物的解离和C3b与C3bBb复合物的解离）来发挥此活性。作为血浆丝氨酸蛋白酶因子I（FI）的辅因子，CR1通过FI增加C4b和C3b的切割，称为辅因子活性（CA），通过抑制C3扩增循环来抑制经典和替代的补体途径，反过来又防止进一步的补体激活。罗杰斯及其同事提供证据表明Aβ肽可以在不存在抗体17的情况下结合并激活补体途径，并表明Aβ肽是cl通过补体依赖于通过RBC 18表达的CR1来循环。 CR1表现出三种类型的多态性:结构或长度多态性，密度多态性，和Knops血型多态性11,19。结构多态性与长同源重复序列（LHR）数量的变化有关，因此定义了四种同工型。事实上，CR1蛋白的细胞外结构域由一系列称为短共识重复（SCR）或补体对照重复（CCP）的重复单元组成。这些SCR已经从编码CR1的补体脱氧核糖核酸（cDNA）证明。 SCRs以七个串联组合排列，称为LHR。 CR1被布置成四个LHRs的，被指定为LHR-A，-B，-C，和-D，从7-SCR单元19，20的重叠所产生的，21 。 以频率增加的顺序，通过蛋白质印迹（WB）测定的这些CR1同种型是CR1 * 1（A / F）（凝胶电泳快速迁移），CR1 * 2（B / S）（凝胶电泳缓慢迁移），CR1 * 3（C / F&#39;）和CR1 * 4（D）。两种最常见的同种型CR1 * 1（A / F）和CR1 * 2（B / S）分别由四个和五个LHR组成，而CR1 * 3（C / F）和CR1 * 4（D ）分别由3和6个LHR组成。由30个SCR组成的最常见的同种型（CR1 * 1）包含三个C4b结合位点（SCR 1-3，8-10和15-17）和两个C3b结合位点（SCR 8-10和15-17） ，而可控硅22-28结合的C1q，纤维胶凝蛋白，和MBL 12，20，21，22，23，24，25。因此，CR1 * 2含有一个额外的C3b / C4b结合位点比较d到CR1 * 1。 图1说明了CR1的四种不同同种型的结构，命名和分子量。 密度多态性对应于表示CR1在RBC上的组成型表达水平的稳定表型。在健康白种人受试者中，已经显示每个RBC的CR1分子的数目可以变化高达10倍（从每个细胞150至1,200个分子） 26 。所述Helgeson表型的RBC具有非常低的CR1密度，这被证明是每个细胞27个 ，28低于150点的分子。 RBCs的CR1密度与CR1基因上的常染色体共显性双重体系遗传相关，与Hin dIII限制性片段长度多态性（RFLP） 29相关 。 CR1基因的内含子27中的单点突变，编码第二个外显子之间LHR-D中的SCR导致在该区域内产生多态性Hin dIII位点30 。的基因组7.4 HindIII位片段和6.9 kDa的分别识别与高（H等位基因）或低（L等位基因）CR1上密度红细胞相关的等位基因。然而，没有相关性的红细胞和HindIII位多态性CR1密度之间在一些西非人群31，32发现。连接CR1密度调节与非编码Hin dIII多态性的机制仍然未知。在几种多态性，Q981H在SCR16和P1786R在SCR28据报道，被链接到CR1密度的红细胞30，33。 根据国际命名法，Knops（KN）多态性是由国际输血学会指定的第22个血型系统。它含有9种抗原特异性CR1对RBCs表达的灵敏度，包括三种抗原抗原KN1 / KN2，KN3 / KN6和KN4 / KN7，以及3种分离的抗原KN5，KN8，KN9。 KN1，KN3，KN4和KN5抗原是KN系统的高频抗原（ 即，在一般群体的99％以上表达）。然而，该多态性在AD中的作用尚待确定13 。 本工作描述的方案旨在确定涉及几种疾病（如AD，系统性红斑狼疮和疟疾）易感性的CR1长度多态性基因型。我们的CR1长度多态性测定方法利用了包含CR1同种型的LHR-B数量和LHR-B和LHR-C之间的序列差异（ 图2 ）。

<table><tbody><tr><td>Lab coat</td><td></td><td></td><td>protection</td></tr><tr><td>SensiCareIce powder-free Nitrile Exam gloves</td><td>Medline Industries, Inc, Mundelein, IL 60060, USA</td><td>486802</td><td>sample protection</td></tr><tr><td>Eppendorf Reference 2 pipette, 0,5-10&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000024</td><td>sample pipetting</td></tr><tr><td>Eppendorf Reference 2 pipette, 20-100&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000059</td><td>sample pipetting</td></tr><tr><td>Eppendorf Reference 2 pipette, 100-1000&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000083</td><td>sample pipetting</td></tr><tr><td>TipOne 10&amp;micro;L Graduated, filter tip</td><td>Starlab GmbH, D-22926 Ahrenburg, Germany</td><td>S1121-3810</td><td>sample pipetting</td></tr><tr><td>TipOne 1-100&amp;micro;L bevelled, filter tip</td><td>Starlab GmbH, D-22926 Ahrenburg, Germany</td><td>S1120-1840</td><td>sample pipetting</td></tr><tr><td>ART 1000E Barrier Tip</td><td>Thermo Fischer Scientific , F-67403 Illkirch, France</td><td>2079E</td><td>sample pipetting</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>0030120086</td><td>mix</td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries, Inc, Bohemia, NY 111716, USA</td><td>SI-0236</td><td>mix</td></tr><tr><td>QIAamp DNA Mini Kit (250)</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>51306</td><td>DNA Extraction</td></tr><tr><td>Buffer AL</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19075</td><td>Lysis buffer</td></tr><tr><td>QIAamp Mini Spin Column</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>1011706</td><td>DNA binding</td></tr><tr><td>Buffer AW1</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19081</td><td>Wash buffer</td></tr><tr><td>Buffer AW2</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19072</td><td>Wash buffer</td></tr><tr><td>Biowave DNA spectrophotometer</td><td>Biochrom Ltd, Cambridge CB4 OFJ, England</td><td>80-3004-70</td><td>DNA concentration&amp;nbsp;</td></tr><tr><td>Mikro 200 centrifuge</td><td>Hettich Zentrifugen, D-78532, Germany</td><td>0002020-02-00</td><td>centrifugation</td></tr><tr><td>Multipette&amp;nbsp;E3</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4987000010</td><td>distribution</td></tr><tr><td>Light Cycler 480 multiwell plate 96, white</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04729692001</td><td>reaction place</td></tr><tr><td>Light Cycler 480 sealing foil</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04429757001</td><td>coverage</td></tr><tr><td>Heraeus Megafuge 11R centrifuge</td><td>Thermo Fischer Scientific , F-67403 Illkirch, France</td><td>75004412</td><td>centrifugation</td></tr><tr><td>LightCycler 480 Instrument II, 96-well</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>05015278001</td><td>high resolution melting polymerase chain reaction</td></tr><tr><td>LightCycler 480 High Resolution Melting Master</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04909631001</td><td>reaction reagents</td></tr><tr><td>light cycler 480 SW 1.5.1 software</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td></td><td>software used for HRM PCR CR1 polymorphism data analysis</td></tr><tr><td>CN3 primer: 5&#039;ggccttagacttctcctgc 3&#039;</td><td>Eurogentec Biologics Division, B- 4102 Seraing, Belgium</td><td></td><td>reaction reagent</td></tr><tr><td>CN3re primer: 5&#039;gttgacaaattggcggcttcg 3&#039;</td><td>Eurogentec Biologics Division, B- 4102 Seraing, Belgium</td><td></td><td>reaction reagent</td></tr></tbody></table>

high-resolution melting pcr for complement receptor 1 length polymorphism genotyping: an innovative tool for alzheimer's disease gene susceptibility assessment

补体受体1（CR1）是在先天免疫系统中发挥关键作用的跨膜糖蛋白，在许多细胞类型，特别是红细胞（RBC）上表达。作为补体成分C3b和C4b的受体，CR1调节补体级联的活化，并促进免疫复合物和细胞碎片以及阿尔茨海默氏病（AD）中淀粉样蛋白β（Aβ）肽的吞噬作用。一些研究已经证实了CR相关的单核苷酸多态性（SNPs）以及拷贝数变异（CNV）。在这里，我们描述了一种确定CR1受体长度多态性的创新方法。受体包括称为长同源重复序列（LHR）-LHR-A，LHR-C和LHR-D的三个结构域，以及n域，LHR-B，其中n为0至3之间的整数。使用单个对的特异性引物，遗传物质用于扩增LHR-B结构域的第一个片段（the变体扩增子B）和LHR-C结构域的第二个片段（不变扩增子）。变体扩增子B和不变扩增子在所述引物的杂交区域外的五个核苷酸处显示差异。使用定量工具（高分辨率融合（HRM）曲线）推导变体扩增子B和不变扩增子的数目，并且变体扩增子B与不变扩增子的比例根据CR1长度多态性而不同。该方法比规范表型方法提供了几个优点，因为它不需要新鲜的材料，并且更便宜，更快，因此适用于较大的群体。因此，使用这种方法应该有助于更好地了解CR1同种型在疾病如AD的发病机制中的作用。

图 6A显示由WB的CR1长度多态性的表型分型。 图 6B和C显示在HRM-PCR分析期间获得的曲线，使得能够确定CR1长度多态性。在来自具有不同CR1同种型的受试者的基因组DNA的PCR衍生的扩增子的高分辨率融合之后使用软件的融合曲线的分析产生根据其同种异型CR1表达表型区分受试者的曲线。 图6显示乙呈现的第一模式中，所谓的"归一化和移熔解曲线"，而图6中 C上显示呈现的第二模式中，被称为"归一化和温度移差异图"。 图6中所显示的表型组分布:（CR1 * 3，* CR1 1）; （CR1 * 1，CR1 * 2）; CR1 * 1; CR1 * 2;和（CR1 * 2，CR1 * 4）。这使得能够使用这种新的分子生物学方法对CR1长度多态性进行基因分型。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56012/56012fig1.jpg" /> 图1: 补体受体1蛋白的结构和多态性的示意图。每个短的共有重复（SCR）或补体对照蛋白（CCP）由一个圆圈表示。 SCR被分组成称为LHR的重复的高阶结构。从胞外区到细胞膜，LHR是:LHR-A，-B，-C，-D和-S（补充或额外的LHR）。在最常见的CR1同种型（CR1 * 1），C4b /衰变加速活性（DAA）结合位点位于LHR-A（SCR 1-3，绿色圆圈），C3b / C4b /辅因子活性（CA）结合位点位于3 LHR-B（SCRs 8-10，红色圆圈）和LHR-C（SCR 15-17，紫色圆圈）的N末端SCR和C1q / MBL和ficolin的结合位点在LHR-D 22-28，蓝色圆圈）。同源结构染色相同。 CR1 * 2（S）同种型提供额外的LHR（LHR-S），因此含有另外的C3b / C4b结合位点。 KDa，千道尔顿NRC，非减少条件。 RC，减少条件。 TM，跨膜结构域。这个数字是从Brouwers 等人改编而来的。 36经自然出版集团许可。 <a href="//ecsource.jove.com/files/ftp_upload/56012/56012fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src=<html> "/files/ftp_upload/56012/56012fig2.jpg"/&gt; 图2 :通过HRM-PCR获得的具有序列扩增子位置的CR1同种型结构的示意图。每个框代表SCR。 SCR分组为LHR:LHR-A，-B，-C和-D。同源结构染色相同。 CR1 * 2和CR1 * 4同种型呈现额外的LHR（LHR-B），因此含有额外的扩增子区域（扩增子B，红色）。 CR1 * 3缺乏LHR-B并且含有较少的扩增子区（缺乏扩增子B）。扩增子B（196bp）和不变扩增子（197bp）之间的核苷酸差异分别描绘为红色和蓝色。通过HRM-PCR测定每个CR1同种型之间的比例差异（扩增子B，红色/不变扩增子，蓝色），使得能够进行基因分型。 CN3和CN3re引物序列用下划线表示。 TM，跨膜结构域。 CYT，细胞质尾巴。pg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/56012/56012fig3.jpg" /> 图3: 用于从人血液样品中对CR1长度多态性进行基因分型的方案的流程图。收集人类DNA样本。提取DNA以获得DNA血液样品。确定DNA浓度，稀释至10 ng /μL的DNA浓度。使用HRM-PCR获得CR1长度多态性基因型。 <a href="//ecsource.jove.com/files/ftp_upload/56012/56012fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56012/56012fig4.jpg" /> 图4 :分光光度计的截图我们<html>以确定DNA浓度。按1（ A ）在分光光度计上选择DNA模式。使用左右箭头（ C ）选择5 mm路径长度（ B ）。使用左右箭头（ C ）选择单位（μg/ mL）（ D ）。使用左右箭头（ C ）选择稀释因子1（ E ）。使用左右箭头选择50（ F ）的因子。按OK （ G ）。将10μL蒸馏水吸入比色皿。将比色皿放入分光光度计（ H ）中。按OA / 100％T按钮（ I ）。移取10μLDNA样品（1/4稀释于蒸馏水中）至比色杯中。将比色皿放入分光光度计（ K ）。按绿色按钮（读/开）（ L ）。注意浓度（ M ）。es / ftp_upload / 56012 / 56012fig4large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/56012/56012fig5.jpg" /> 图5: 协议步骤4中使用的软件的图形界面截图。 （ A ）打开基因扫描软件。 （ B ）扩增程序和熔解曲线程序。 （ C ）单击模块栏中的样本编辑器 。 （ D ）选择扫描 。 （ E ）定义样品的性质。 （ F ）单击模块栏中的分析 。 （ G ）单击" 归一化"选项卡以对熔解曲线进行归一化。 （ H ）单击温度移位选项卡以显示两者的熔解曲线归一化和温度偏移。 （ I ）单击" 计算"按钮分析结果并确定分组。 （ J ）点击差异绘图选项卡以查看归一化和转移熔融曲线以及归一化和温度移位差分图 。 （ K ）根据CR1长度基因型对样品进行彩色分组。 <a href="//ecsource.jove.com/files/ftp_upload/56012/56012fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/56012/56012fig6.jpg" /> 图6: 通过HRM-PCR获得的WB观察到的CR1长度多态性及其相应的轮廓曲线的表型。 （ A ）使用WB对CR1长度多态性进行表型分析。 （ B </strong&gt;）和（ C ）使用HRM-PCR分析的CR1长度多态性的基因分型。从显示不同CR1同种型的受试者的基因组DNA获得的PCR扩增子的HRM曲线分析导致鉴定特异性曲线:（CR1 * 3，CR1 * 1）（紫色）; （CR1 * 1，CR1 * 2）（蓝色）; CR1 * 1（绿色）; CR1 * 2（红色）;和（CR1 * 2，CR1 * 4）（灰色）。这些对应于CR1长度多态性等位基因。如表2所示的"归一化和转移熔融曲线"（B）和"归一化和温差曲线图"（C）所示，曲线分布根据（CR1 * 3，CR1 * 1） ; （CR1 * 1，CR1 * 2）; CR1 * 1; CR1 * 2;和（CR1 * 2，CR1 * 4）组。这个数字改编自Mahmoudi et al。 38经Elsevier许可。 <a href="//ecsource.jove.com/files/ftp_upload/56012/56012fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessmen

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

在这里，我们描述了一种用于确定补体受体1（CR1）长度多态性的创新方法，用于多种应用，特别是对诸如阿尔茨海默病（AD）等疾病的易感性的评估。这种方法可能有助于更好地了解CR1同种型在AD发病机制中的作用。

高分辨率融合PCR用于补体受体1长度多态性基因分型:阿尔茨海默病基因易感性评估的创新工具

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

high-resolution-melting-pcr-for-complement-receptor-1-length

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

11.7K 次观看.  Reims University Hospitals, Robert Debré Hospital. 在这里，我们描述了一种用于确定补体受体1（CR1）长度多态性的创新方法，用于多种应用，特别是对诸如阿尔茨海默病（AD）等疾病的易感性的评估。这种方法可能有助于更好地了解CR1同种型在AD发病机制中的作用。 

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

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

科研

神经科学

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.

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.

血栓烷 A2 受体基因 C924T 多态性的研究方法

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

遗传学

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

利用染色质配置的计算分析，将阿尔茨海默氏病变异体映射到其目标基因

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome&#8211;negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

使用高分辨率熔解分析检测 CALR 基因中的遗传变异

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

免疫与感染

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

广义心理相互作用 (PPI) 分析阿尔茨海默病遗传危险个体的记忆相关连通性

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

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.

遗传性 Transthyretin Ala97Ser 相关淀粉样变性的遗传分析

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

医学

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...