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Multiplexed, High-Throughput Genotyping by Single-Base Extension and End-Labeled Free-Solution Electrophoresis
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Section:Abstract
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.
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This article has been cited by 13 ACS Journal articles (5 most recent appear below).
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Jishan Li and Wenwan ZhongAnalytical Chemistry2007 79 (23), 9030-9038Typing of Multiple Single-Nucleotide Polymorphisms by a Microsphere-Based Rolling Circle Amplification Assay
Jishan Li and Wenwan ZhongAnalytical Chemistry2007 79 (23), 9030-9038The combination of suspension array with rolling circle amplification can lead to a sensitive and specific assay for single-nucleotide polymorphisms (SNPs) detection, as demonstrated in this study. A circular template generated by ligation upon the ...
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History
- Published In Issue September 01, 2002
- Received for review May 28, 2002. Accepted July 2, 2002.
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