Medium-Throughput SNP Genotyping Using Mass Spectrometry: Multiplex SNP Genotyping Using the iPLEX® Gold Assay
Medium-Throughput SNP Genotyping Using Mass Spectrometry: Multiplex SNP Genotyping Using the iPLEX® Gold Assay
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DOI：
10.1007/978-1-61737-954-3_5
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Millis, Meredith P.
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作者：
Millis, Meredith P.
根据研究项目的范围，单核苷酸多态性（SNP）基因分型实验的类别从低到中到高通量不等，每种方法在成本，平台和效率方面都有很大差异。中等通量基因分型通常适用于在384个个体中检测多达36个标记，并且通常用于在基因组扫描中识别的染色体区域的精细定位。多路复用，允许同时评估多个SNP，是一种有效，快速和经济的方式来增加中通量基因分型输出，并很容易使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行。在本章中，我们描述了一种使用Sequenom公司提供的iPLEX（R）Gold检测试剂盒进行中等通量基因分型的技术。(San Diego，CA）。该多重SNP基因分型平台包括基因组DNA的位点特异性PCR扩增，随后是虾碱性磷酸酶处理以去除未掺入的核苷酸，使用质量修饰的终止子进行单碱基引物延伸，以及用于等位基因特异性检测的MALDI-TOF MS。该方案利用专有的酶、软件和SpectroCHIP（R）阵列，这些阵列用MALDI矩阵预先点样。
Depending on the scope of the research project, categories of single-nucleotide polymorphism (SNP) genotyping experiments range from low to medium to high throughput, with each approach differing widely in cost, platform, and efficiency. Medium-throughput genotyping is generally appropriate for assaying up to 36 markers in 384 individuals and is commonly used for fine-mapping chromosomal regions identified in genome scans. Multiplexing, which allows for simultaneous assessment of multiple SNPs, is an efficient, rapid, and economic way to augment medium-throughput genotyping output and is readily performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this chapter, we describe a technique for medium-throughput genotyping using the iPLEX (R) Gold assay available from Sequenom, Inc. (San Diego, CA). This multiplex SNP genotyping platform incorporates locus-specific PCR amplification of genomic DNA, followed by shrimp alkaline phosphatase treatment to inactivate unincorporated nucleotides, single-base primer extension using mass-modified terminators, and MALDI-TOF MS for allele-specific detection. This protocol utilizes proprietary enzymes, software and SpectroCHIP (R) Arrays that are pre-spotted with a MALDI matrix.