Vreeland Wyatt N, Meagher Robert J, Barron Annelise E
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208-3120, USA.
Anal Chem. 2002 Sep 1;74(17):4328-33. doi: 10.1021/ac0258094.
Technologies that allow for high-throughput, economical, and accurate single nucleotide polymorphism (SNP) genotyping are becoming crucial for modern genomic efforts. Here, we present a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). Three unique SBE oligonucleotide primers, which probe for mutations of clinical importance in the human p53 gene, were covalently conjugated to three unique polypeptoid frictional end labels and mixed together. This primer-polypeptoid conjugate cocktail was then used in a multiplexed SBE reaction followed by free-solution separation in a 96-capillary array electrophoresis (CAE) instrument. The study was designed to demonstrate multiplexed SNP genotyping of several loci in a single reaction and a single subsequent analysis. Further, the electrophoretic analysis was conducted without any viscous polymeric separation media, was complete in less than 10 min, and can be implemented in any capillary or microfluidic electrophoretic system with four-color fluorescent detection capabilities. Multiplexed SBE-ELFSE genotyping analysis resulted in the simultaneous and accurate genotyping of three p53 loci on five different DNA templates in a single reaction set and single CAE analysis. With the implementation of this method in 96 or more capillaries in parallel, high-throughput screening of SNPs will be accessible to a large number of laboratories.
能够实现高通量、经济且准确的单核苷酸多态性（SNP）基因分型的技术，正成为现代基因组研究的关键。在此，我们介绍一种多重单碱基延伸（SBE）基因分型方法，该方法利用了通过末端标记自由溶液电泳（ELFSE）实现的独特分离模式。三种独特的SBE寡核苷酸引物，用于探测人类p53基因中具有临床重要性的突变，它们与三种独特的聚肽摩擦末端标记共价结合并混合在一起。然后，将这种引物 - 聚肽共轭物混合物用于多重SBE反应，随后在96毛细管阵列电泳（CAE）仪器中进行自由溶液分离。该研究旨在证明在单个反应和单个后续分析中对多个位点进行多重SNP基因分型。此外，电泳分析无需任何粘性聚合物分离介质，在不到10分钟内即可完成，并且可以在任何具有四色荧光检测能力的毛细管或微流控电泳系统中实施。多重SBE - ELFSE基因分型分析在单个反应组和单个CAE分析中，实现了对五个不同DNA模板上的三个p53位点的同时准确基因分型。通过在96根或更多毛细管中并行实施该方法，大量实验室将能够进行SNP的高通量筛选。
