Name: a robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in fragile x mental retardation-1 gene
Uploaded: 2019-09-16T13:00:00
Description: Watch this Scientific Journal Video about A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene at JoVE.c

这项研究得到了国家自然科学基金应急管理项目（第81741004号赠款）、国家自然科学基金（第81860272号）、广西省科技基金重大研究计划（第81741004号）的资助。授予号AB16380219），中国博士后科学基金资助（批号2018M630993）和广西自然科学基金（批号2018GXNSFAA281067）。

香港中文大学-新界东集群临床研究伦理委员会获伦理批准（参考编号：2013.055）
1. PCR扩增
<ol><li>开始之前，从-20°C冷冻室中取出PCR缓冲液混合物、样品稀释剂和DNA样品（包括测试和参考DNA）（参见材料表），并将其保持在室温下20-30分钟，以确保所有试剂和DNA完全解冻。涡旋，并在使用前短暂旋转。</li>
	<li>使用分光光度计测量DNA样品的浓度（见材料表）?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+premutations+in+familial+premature+ovarian+failure.">Fragile X premutations in familial premature ovarian failure.</a> Lancet. 346, 309-310 (1995).</li><li>Van Esch, H., Buekenhout, L., Race, V., Matthijs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+early+premature+ovarian+failure+in+two+sisters+compound+heterozygous+for+the+FMR1+premutation.">Very early premature ovarian failure in two sisters compound heterozygous for the FMR1 premutation.</a> European Journal of Medical Genetics. 52, 37-40 (2009).</li><li>Bourgeois, 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=A+review+of+fragile+X+premutation+disorders:+expanding+the+psychiatric+perspective.">A review of fragile X premutation disorders: expanding the psychiatric perspective.</a> Journal of Clinical Psychiatry. 70, 852-862 (2009).</li><li>Farzin, 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=Autism+spectrum+disorders+and+attention-deficit/hyperactivity+disorder+in+boys+with+the+fragile+X+premutation.">Autism spectrum disorders and attention-deficit/hyperactivity disorder in boys with the fragile X premutation.</a> Journal of Developmental and Behavioral Pediatrics. 27, S137-S144 (2006).</li><li>Hantash, F. 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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=FMR1+allele+size+distribution+in+35,000+males+and+females:+a+comparison+of+developmental+delay+and+general+population+cohorts.">FMR1 allele size distribution in 35,000 males and females: a comparison of developmental delay and general population cohorts.</a> Genetics in Medicine. 20 (12), 1627-1634 (2018).</li><li>Saul, R. A., Tarleton, J. 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W., Morgan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+carriers+of+premutation-size+alleles+of+the+FMRI+gene--and+implications+for+the+population+genetics+of+the+fragile+X+syndrome.">Prevalence of carriers of premutation-size alleles of the FMRI gene--and implications for the population genetics of the fragile X syndrome.</a> American Journal of Human Genetics. 57, 1006-1018 (1995).</li><li>Tassone, 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=FMR1+CGG+allele+size+and+prevalence+ascertained+through+newborn+screening+in+the+United+States.">FMR1 CGG allele size and prevalence ascertained through newborn screening in the United States.</a> Genome Medicine. 4, 100 (2012).</li><li>Dombrowski, 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=Premutation+and+intermediate-size+FMR1+alleles+in+10572+males+from+the+general+population:+loss+of+an+AGG+interruption+is+a+late+event+in+the+generation+of+fragile+X+syndrome+alleles.">Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.</a> Human Molecular Genetics. 11, 371-378 (2002).</li><li>Cronister, A., Teicher, J., Rohlfs, E. 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FXS是仅次于三联体21的第二大智力障碍原因，占X相关智力迟钝30的近一半，这可能影响大约四千名男性中的一个和八千名女性中的一个。更重要的是，近1在250-1，000女性携带预突变，这个频率是1在250-1，600男性26，31，32，33。由于 CGG 重复扩张到完全突变的风险在将预突变等位基因传?...

易碎 X 综合征 （FXS） 和易碎 X 相关疾病，例如震颤和失流综合征 （FX-TAS），以及原发性卵巢功能不全 （FX-POI） 主要是由 5' 未翻译的细胞氨酸-瓜氨酸 -瓜氨酸 （CGG） 三核苷酸重复扩张引起的Xq27.31，2. 脆弱 X 智力迟钝 -1（FMR1）基因的区域 （UTR）FMR1编码蛋白（FMRP）是一种多核体相关的RNA结合蛋白，通过调节mRNA的替代拼接、稳定性和树突状传输或调制合成，在神经元发育和突触可塑性方面发挥作用部分贴午后蛋白3，4，5，6，7。
CGG重复大小为>200的动态变异被描述为完全突变，导致FMR1启动子8的异常超甲基化和随后转录沉默。由此产生的FMRP蛋白的缺失或缺乏会破坏正常的神经元发育，并导致FXS9，其特征是各种临床症状，包括中度至重度智力残疾、发育迟缓、过度活跃行为、不良接触和孤独症表现10，11，12。女性FXS患者的表达通常比男性温和。CGG 重复大小从 55 到 200 和 45 到 54 分别归类为预突变和中间状态。由于高度不稳定，CGG重复大小在预突变或中间等位基因大概扩大时，从父母传给后代13，14。因此，具有预突变等位基因的携带者由于重复扩张而使儿童受FXS影响的风险很高，在某些情况下，中间等位基因可以将其重复大小扩展到两代15之间的全部突变范围。 16.此外，预变异的男性也增加患晚发病FX-TAS17、18、19的风险，而预突变女性则倾向于FX-TAS和FX-POI20， 21，22.最近，有报道称，患有发育迟缓和社会行为问题的自闭症谱系障碍在预突变FMR1等位基因23、24的儿童中出现。
确定确切的CGG重复大小对于FXS和易碎X相关疾病的分类和预测具有重要意义25，26。从历史上看，CGG重复区域特异性聚合酶链反应（PCR）具有片段大小加上南方斑点分析，是FMR1 CGG重复27分子分析的黄金标准。然而，传统的特异性PCR对超过100至130次重复的大型预突变不太敏感，并且无法扩增完全突变27，28。此外，用于重复尺寸的传统基因分析仪上的毛细血管电泳无法检测超过 200 CGG 重复的FMR1 PCR 产品。南方印斑分析能够区分更广泛的重复大小，从正常突变到完全突变重复数，并已广泛用于确认完全突变（在男性）和区分异质异位基因与完全突变明显具有正常重复大小的同源性等位子（在女性中）。但是，量化重复的分辨率是有限的。更重要的是，这种分步测试策略是劳动密集型、耗时且成本效益高的。
在这里，我们提出了一种准确和可靠的基于PCR的方法，用于定量FMR1的CGG重复。本测试的第一步是使用易碎 X PCR 试剂盒对FMR1启动器的 5'UTR 中的重复序列进行 PCR 扩增。PCR 产品经过纯化，片段大小在微流体毛细管电泳仪器上执行，随后使用分析软件对 CGG 重复次数进行解释，通过引用基于PCR 片段长度与 CGG 重复次数成正比。PCR系统包括促进高GC富分子三核苷酸重复区域扩增的试剂。这种基于PCR的测定是可重复的，能够识别FMR1启动子的所有CGG重复范围。这是一种经济高效的方法，可在FXS和易碎X相关疾病的分子诊断和筛查中得到广泛应用，周转时间少，设备投资更少，因此可用于更广泛的临床实验室。

<table>
	<tbody>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: 0.2 mL PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02D-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: 1X TE buffer, pH 8.0, Rnase-free</td>
			<td>Ambion</td>
			<td>AM9849</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: 2100 Bioanalyzer instrument</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: 96-well PCR Plate</td>
			<td>Thermo Fisher</td>
			<td>AB0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: Electrode cartridge</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies equipment of the 2100 Bioanayzer instrument</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: IKA vortex mixer</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies equipment of the 2100 Bioanayzer instrument</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: Sizing software 2100 Expert software</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies equipment of the 2100 Bioanayzer instrument</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument: Test chips</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies equipment of the 2100 Bioanayzer instrument</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit</td>
			<td>Agilent</td>
			<td>5067-1506</td>
			<td>For Fragment sizing</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: DNA 7500 Ladder (yellow cap)</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: DNA 7500 Markers (green cap)</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: DNA chips</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: DNA Dye Concentrate (blue cap)</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: DNA Gel Matrix Vial (red cap)</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: Electrode Cleaner</td>
			<td>Agilent</td>
			<td></td>
			<td>In kit: Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: Spin Filter</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 kit: Syringe</td>
			<td>Agilent</td>
			<td></td>
			<td>Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506)</td>
		</tr>
		<tr>
			<td>Chip priming station</td>
			<td>Agilent</td>
			<td>5065-4401</td>
			<td>Supplies equipment of the 2100 Bioanayzer instrument</td>
		</tr>
		<tr>
			<td>Cubee Mini-centrifuge</td>
			<td>GeneReach</td>
			<td>aqbd-i</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter plate vacuum Manifold: MultiScreenHTS Vacuum Manifold</td>
			<td>Merck Millipore</td>
			<td>MSVMHTS00</td>
			<td>Vacuum instrument for Filter plate vacuum Manifold for PCR product purification</td>
		</tr>
		<tr>
			<td>Filter plate vacuum Manifold: Silicone stopper</td>
			<td>Merck Millipore</td>
			<td>XX2004718</td>
			<td>Filter plate vacuum Manifold</td>
		</tr>
		<tr>
			<td>Filter plate vacuum Manifold: Vacuum pump</td>
			<td>Merck Millipore</td>
			<td>WP6122050</td>
			<td>Filter plate vacuum Manifold</td>
		</tr>
		<tr>
			<td>Filter plate vacuum Manifold: Waste collection vessel</td>
			<td>Merck Millipore</td>
			<td>XX1004705</td>
			<td>Filter plate vacuum Manifold</td>
		</tr>
		<tr>
			<td>FragilEase Fragile X PCR kit</td>
			<td>PerkinElmer</td>
			<td>3101-0010</td>
			<td>For PCR amplification</td>
		</tr>
		<tr>
			<td>FragilEase Fragile X PCR kit: Sample Diluent</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 )</td>
		</tr>
		<tr>
			<td>FragilEase PCR Buffer mix</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ), containing primers. Primer sequences: TCAGGCGCTCAGCTCCGTTTCGGTTTCA (forward) 
			FAM-AAGCGCCATTGGAGCCCCGCACTTCC (reverse)</td>
		</tr>
		<tr>
			<td>FragilEase Polymerase</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 )</td>
		</tr>
		<tr>
			<td>FraXsoft analysis software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-2000 Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR clean up plate: NucleoFast 96 PCR plate</td>
			<td>MACHEREY-NAGEL</td>
			<td>743100</td>
			<td></td>
		</tr>
		<tr>
			<td>reference DNA sample</td>
			<td>Coriell</td>
			<td>NA20240 &#38; NA20239</td>
			<td></td>
		</tr>
		<tr>
			<td>S1000 96-well Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1852196</td>
			<td>This can be replaced by other Thermal Cyclers (eg. Veriti&#8482; 96-Well Thermal Cycler, Applied Biosystems, catalog number: 4375786)</td>
		</tr>
		<tr>
			<td>TriNEST Incubator/Shaker instrument</td>
			<td>PerkinElmer</td>
			<td>1296-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Life Technologies</td>
			<td>10977015</td>
			<td>For 2100 Bioanalyzer electrode cleaning</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-0256 (Model G560E)</td>
			<td>Conventional vortex mixer</td>
		</tr>
	</tbody>
</table>

a robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in fragile x mental retardation-1 gene

易碎 X 综合征 （FXS） 和相关疾病是由易碎 X 智力迟钝-1（FMR1）基因启动子的 5' 未翻译区域 （UTR） 中细胞氨酸-瓜氨酸 -鸟-鸟苷 （CGG） 三核苷酸重复的扩展引起的。按照惯例，基因分析仪上的毛细血管电泳片段分析用于FMR1的CGG重复大小，但当重复数高于200时，需要额外的南方印斑分析才能进行精确测量。在这里，我们提出了一种准确和强大的聚合酶链反应（PCR）为基础的方法，用于定量FMR1的CGG重复。本测试的第一步是使用易碎 X PCR 试剂盒对FMR1启动子的 5'UTR 中的重复序列进行 PCR 扩增，然后对 PCR 产品进行纯化和微流体毛细管电泳仪器上的片段大小，以及随后通过使用分析软件引用已知重复项的标准来解释 CGG 重复次数。这种基于PCR的测定是可重复的，能够识别FMR1启动子的全部CGG重复，包括重复数超过200（分类为完全突变）、55至200（预突变）、46至54（中间）和10至45（正常）。它是一种经济高效的方法，有助于对 FXS 和易碎 X 相关疾病进行分类，具有鲁棒性和快速报告时间。

预突变母参考样本（NA20240，重复尺寸30和80）和全突变母参考样本（NA20239，重复尺寸为20和200）的大小调整结果分别如图1A和图1B所示。通常，片段大小配置文件中包括两个标记峰（下标记 50 基对 [bp] 和上标记 10，380 bp）。通常有一个底漆复合峰，其大小接近 95 bp。通过参考样本，可以构造一个包含四个点的线性回归标准曲线，如图1C</st...

Watch this Scientific Journal Video about A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene at JoVE.c

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

精确和强大的聚合酶链反应测定，用于量化易碎 X 智力迟钝-1 基因中细胞氨酸-瓜氨酸-鸟-鸟三核苷酸重复，有助于分子诊断和筛选易碎 X 综合征和易碎 X 相关周转时间较短，设备投资时间缩短。

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; ...

这种经济高效的方法在FXS和易碎X相关疾病的分子诊断和筛选中具有广泛的应用。周转时间快，设备投资少。我们基于 PCR 的测定可促进各种 FXS 和脆弱 X 相关疾病的分类，包括中间、预突变和完全突变，具有鲁棒性和快速报告时间。来自新加坡珀金埃尔默的蔡文将演示这一程序。首先从 20 摄氏度的冰柜中取出 PCR 缓冲液混合物、样品稀释剂和 DNA 样品，并在室温下解冻 20 至 30 分钟。在使用试剂之前，涡流并短暂旋转。使用分光光度计测量DNA样品浓度，如有必要，用样品稀释剂稀释至每微升25纳米。标记 PCR 板的孔，以识别参考和测试的 DNA 样本，并计算测试、参考和阴性控制样本所需的反应数。通过将 15 微升 PCR 缓冲液混合到 0.6 微升样品稀释剂和 0.4 微升聚合酶，准备 PCR 主混合物。漩涡混合和旋转下来。然后将混合物的18微升倒入每井中。然后移液将每个DNA的两个微升放入适当的井中。通过上下移液五次混合试剂，然后密封板。将板放在热循环器中，然后根据手稿指示运行 PCR。将孵化器摇床预热至 65 摄氏度，并将 80 微升 1X TE 缓冲液添加到每个 PCR 产品中。使用多通道移液器将样品转移到 PCR 清理板，然后将板放入摇床中。在 65 摄氏度下孵育，同时在 1200 RPM 下摇晃 10 分钟。孵育后，将孵化器摇床冷却至25摄氏度，将真空仪器设置为250毫巴，并通过过滤器吸气溶液。15分钟后，油井应没有剩余液体。关闭真空，向每井添加 50 微升 1X TE 缓冲液。使用以前的真空设置吸化解决方案 10 分钟。将滤盘底部用力压在一叠纸巾上，然后将 20 微升 1X TE 缓冲液添加到每个井的底部中心，以干燥滤板的底部。将板放在摇床中，在 25 摄氏度下孵育，同时在 1200 RPM 下摇晃 5 分钟。孵育后，将净化PCR产品的至少15微升转移到新鲜的96井PCR板中。在开始之前，将DNA染料浓缩物、DNA凝胶基质、DNA标记、DNA梯和纯化DNA样本带到室温。通过更换注射器并调整底板来设置吸油站，然后松开注射器夹的操纵杆，将其滑到顶部位置。启动尺寸调整软件并准备凝胶染料混合物。涡流染料浓缩10秒，然后旋转下来。然后，将25微升染料加入凝胶基瓶，并涡旋溶液混合。将凝胶染料混合物转移到旋转过滤器中，将其放在离心机中，并在 1500 倍 g 下旋转 10 分钟。当准备加载凝胶染料混合物时，将新的 DNA 芯片插入注油站，并将 9 微升的凝胶染料混合物加入标有 G.Close 的注油站，并确保柱塞位于一毫升标记的位置。按下注射器柱塞，直到夹夹按住。等待整整 30 秒，然后释放剪辑。再等五秒钟，然后慢慢将柱塞拉回一毫升的位置。打开注油站，在标有 G.的井中加入 9 微升凝胶染料混合物，在标有梯子符号的井中加入 5 微升标记，并在 12 个样品井中各加入一个。将梯子的一微升添加到井中，带梯子符号，将 PCR 产品或水的微升添加到样品井中。在 2400 RPM 转速下旋转芯片一分钟。将其插入生物分析器，并在五分钟内运行芯片。运行完成后，将峰值数据导出为 csv 表文件。预突变母样品和全突变母样品用作参考样本。片段大小剖面中包括上部和下标记峰，并且通常有一个底向复合峰值，大约为 95 个基对。这些参考样本用于构造回归标准曲线。然后使用线性回归标准曲线计算未知样本的重复大小。临床法、中间、预突变、全突变、马赛克全突变样本可用这种方法正确分类。在某些情况下，微流体电泳结果中只显示一个峰值，这很可能是由于存在正常同源等位基因。一种次优结果是基线偏差，可能不明确或不可解释，并且被怀疑是由仪器故障引起的。在尝试此程序时，在开始协议之前，确保适当数量的DNA和质量非常重要。FMR1基因测序可以进行，以确定AGG中断模式。和甲基化敏感限制酶或二硫化物修饰测定可用于监测甲基化状态。这是一种快速、可靠且经济高效的技术。它将帮助其他研究人员进行基于人群的关于FXS和脆弱X相关疾病的载体状态的研究。

Research

JoVE Journal

Genetics

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

科研

遗传学

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Quantifying Abdominal Pigmentation in Drosophila melanogaster

量化腹部色素沉着<em&gt;黑腹果蝇</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

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

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

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

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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

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 ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Manipulation of Gene Function in Mexican Cavefish

墨西哥鱼基因功能的操纵

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...