High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates

Abstract

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates

“…ET cassette primer-labeling might now be used in assays where multiplexing and high sensitivity are required, including microarray hybridization formats, other DNA based assays, as well as protein and cell labeling [8,60,66,67]. Additionally, the ability to label any sequencing primer with an ET cassette label will facilitate custom sequencing methods and plasmid vector formats beyond those containing the universal M13 sequences.…”

“…Additionally, the practical utility and multicolor detection capability of the radial mCAE platform was demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel in , 100 s using a noncovalent 2-color staining method [10]. Noncovalent staining with bis-intercalating dyes is reliant on nondenaturing sieving matrices such as hydroxyethylcellulose (HEC), which limits resolution to 5-10 bp [10,66]. Moreover, this labeling scheme requires careful sample quantitation and dye incubation [10].…”

“…The feasibility of using allele-specific PCR with covalently labeled primers for high-speed fluorescent SNP typing on a mCAE wafer was demonstrated by typing the S65C polymorphism associated with HHC in a Swiss population [66]. This format reduces sample generation to a single step and eliminates the need for intercalating dyes.…”

“…This format reduces sample generation to a single step and eliminates the need for intercalating dyes. However, resolution was still limited by the use of HEC as the separation matrix and it was found that a large concentration of PCR product was needed for detecting 6-carboxy-X-rhodamine-(ROX)-labeled amplicons due to its poor fluorescent excitation at 488 nm [66].…”

“…However, the same resources have not been available for robust batch analysis of mCAE generated genotyping data. Indeed, many previous post-run analyses involved processing with several different software programs, so the time needed for data analysis far exceeded the separation time [10,11,19,66,100]. Our approach to solving this problem has focused on adapting robust commercial software for mCAE genotyping analysis [100].…”