Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry
Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry
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DOI：
10.1093/nar/gnf084
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发表时间：
2002-08-15
影响因子：
14.9
通讯作者：
Ju, JY
中科院分区：
文献类型：
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作者：
Kim, S;Edwards, JR;Ju, JY
我们报告了一种采用固相可捕获的生物素化二脱氧核苷酸（生物素- ddntps）进行单碱基扩展的方法，通过质谱（MS）进行多重基因分型。在该方法中，DNA聚合酶用生物素- ddntps扩展具有不同分子量且特异性于DNA模板中多态性位点的寡核苷酸引物，生成3'-生物素化DNA产物。这些产物随后被链霉亲和素包覆的固相磁珠捕获，而未延伸的引物和反应中的其他成分则被冲走。随后将纯延伸DNA产物从固相中释放出来，用基质辅助激光解吸/电离飞行时间质谱法分析。延伸产物的质量用稳定的寡核苷酸作为共同的内部质量标准来测定。由于只有纯延伸DNA产物被引入MS进行分析，因此所得质谱中没有非延伸引物峰及其相关二聚体，这增加了单核苷酸多态性（SNP）分析的准确性和多路复用的范围。固相纯化方法还有助于捕获的寡核苷酸的脱盐，这对于ms精确的质量测量至关重要。我们选择了四种分子量不同的生物素-ddNTPs来产生延伸产物，与传统的ddNTPs相比，其质量差增加了2倍。这种质量差的增加提高了在质谱中检测杂合子的分辨率和准确性。利用这种方法，我们同时区分了模拟p53基因突变的合成DNA模板上的6个核苷酸变异和人类遗传性血色素沉着症基因中2个与疾病相关的snp。
We report an approach using solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3'-biotinylated DNA products. These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to the MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS. We selected four biotin-ddNTPs with distinct molecular weights to generate extension products that have a 2-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum. Using this method, we simultaneously distinguished six nucleotide variations on synthetic DNA templates mimicking mutations in the p53 gene and two disease-associated SNPs in the human hereditary hemochromatosis gene.