Name: candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry
Uploaded: 2018-06-21T12:30:00
Description: Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

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本文所用的遗传物质在伦理上被批准用于儿童办公室 HREC (人类研究伦理学委员会) (CDF/07/492)、人类服务 HREC 部 (10/07) 和皇家儿童医院 HREC (27047)。
1. 设计复用试验
<ol><li>准备一个 SNP 列表进行化验设计。<ol>
<li>将目标区域输入到 Haploview (https://www.broadinstitute.org/haploview/downloads) 的标注器函数中。使用 LD (相关性, r2) 之间的 snp 跨越目标区域, 以生成一个 snps 列表, 它提供?...

<ol>
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在这里, 我们演示了用质谱复用基因分型的方法。用 PCR 配对的 MALDI 质谱4与材料表13中所列的化学分析结果进行了研究。在这个平台上, 我们在实验室的40小时内共生成了11295个基因型, 1255 个个体9个 snp。
我们通过在发现临床队列 phenotyped 对食物过敏和独立复制的候选基因IL13中生成和分析 SNP 数据, 说明该技术在回答遗传?...

在人类复杂的疾病中, 遗传变异有助于疾病的易感性和量化这些变种可能有助于了解发病机制, 识别高危患者组和治疗应答者。的确, 精确医学的承诺依赖于利用基因组信息来识别患者亚群1。不幸的是, 在复杂的疾病生物学空间中, 疾病表型的基础是大量的遗传异质性、低显性和可变表现力, 在基因组范围内识别小说的队列大小要求候选人往往是令人望而却步的大2。另外, 有针对性的候选基因方法首先是关于疾病病因学3中特定基因/通路的先验假说。通路分析工具通常用于调查确定的靶位点的病理生理学, 从而产生许多候选通路。我们在这里演示了一个多重基因分型方法, 允许调查数以万计的 snp 与一个化验, 适合人类队列研究4。这种方法是相对较高的通过投入, 允许成百上千的 DNA 样本被 genotyped 新的发现研究和研究的特定途径。这里概述的方法是有用的识别风险等位基因及其与临床特征的关联, 以相对快速和廉价的方式。该平台为5、6和最近的微生物感染7和人乳突病毒8的筛查和诊断目的提供了极大的优势。
本议定书首先从选择一组基因进行调查, 即目标区域, 通常是通过文献检索确定的, 或者是对参与疾病过程的先验假说;也可能被选为复制, 作为一个发现基因组范围的协会 (GWA) 研究的主要协会。从基因组, 研究员将选择一个精致的标签 SNPs 列表。即, 在该区域的变种之间的连锁不平衡 (LD) 或相关性, 用来确定一个代表的 "标记 snp" 的一组 snp 在高 LD, 称为单体。该区域的高 LD 意味着, snp 经常被遗传在一起, 这样, 基因型一 SNP 就足以代表单体中所有 SNPs 的变异。或者, 如果对来自许多地区的 SNPs 的明确列表进行跟踪, GWA 研究的复制, 这个过程可能是不必要的。对于复用基因分型, 必须围绕这些目标设计一个化验, 以便放大引物与延伸底漆和产品的质量不同, 以产生解释质谱。这些参数很容易实现的多重基因分型分析设计工具。该设计的正反引物将被用来瞄准感兴趣的标记并放大包含 SNP 的序列。引伸引物直接附着在 SNPs 上, 并添加了一个与 snp 互补的单质量修饰的 "终结者" 基础。终止者基地防止进一步扩展脱氧核糖核酸。该基的质量修正使不同的碎片由一个单一的基地被检测到质谱。将含有基因分型化学的板块应用于一种用于质谱平台测量的芯片。在对系统检测到的原始基因分型调用应用适当的质量控制后, 可以导出数据并用于统计分析, 以测试与疾病表的相关性。

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA&#160;</td>
			<td style="width:127px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">1 &#956;L at a concentration of 5-10 ng/&#956;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primers: forward and reverse amplification and extension</td>
			<td style="width:127px;">IDT</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">see manuscript section 1.2.1 on design of primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water&#160;</td>
			<td style="width:127px;">E.g. Milli-Q water&#160;</td>
			<td style="width:119px;">-</td>
			<td>deionized with 18.2 M&#937;.cm resistivity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Genotyping reagent kit. iPLEX Gold Chemistry reagent set&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;">#10148-2</td>
			<td style="width:235px;">includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR plates (384-well)</td>
			<td style="width:127px;">Abgene</td>
			<td style="width:119px;">#ABGAB-1384</td>
			<td style="width:235px;">For the MassARRAY system plates by Abgene are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipettes</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>single and 8-channel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>compatible with 384-well plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermocycler</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;">compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimple resin plate&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;"></td>
			<td>6mg, 384-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate rotator&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MassARRAY Analyzer 4 System</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">MALDI-TOF (matrix-assisted laser desorption/ionization &#8211; time of flight) Mass Spectrometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RS1000 Nanodispenser</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Assay Design Suite</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Tool used to design the multiplex genotyping assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Start Taq</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>DNA polymerase enzyme&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin&#160;</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Supplied with iPLEX kit</td>
		</tr>
	</tbody>
</table>

candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry

复杂的疾病往往是由多种常见的遗传变异所支撑的, 它们导致了疾病的易感性。在这里, 我们描述了一个经济高效的标签单核苷酸多态性 (SNP) 方法, 使用多路基因分型检测与质谱, 以调查基因通路协会的临床团体。我们研究食品过敏候选者轨迹Interleukin13 (IL13) 作为一个例子。此方法利用区域内的共享链接不平衡 (LD) 有效地最大化覆盖率。然后将所选的 LD snps 设计成多路复用检测, 使多达40种不同的 snps 同时进行分析, 提高成本效益。聚合酶链反应 (PCR) 用于放大目标基因座, 其次是单核苷酸扩展, 然后用基质辅助激光解吸/电离时间 (MALDI) 质谱测量 amplicons。用基因型呼叫软件对原始输出进行分析, 采用严格的质量控制定义和截割方法, 确定高概率基因型, 并输出数据分析。

通过上述的协议, 我们 genotyped 标记 snp 横跨 Th2 免疫基因IL13在一组食物过敏病例和控制9。我们应用了逻辑回归分析, 调整了祖先和其他潜在的变量, 以测试是否有兴趣区域内的遗传变异增加了食品过敏风险。表 109显示一个变种 rs1295686 与挑战被证明的食物过敏相关, 并且我们在复制队列证实了这个协会使用同样复用基?...

Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

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

鉴定导致复杂人类疾病的遗传变异使我们能够确定新的机制。在这里, 我们展示了一个多重基因分型方法的候选基因或基因通路分析, 最大限度地扩大覆盖率, 以低成本, 并服从队列为基础的研究。

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

Research

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在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

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

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