Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis

Abstract

In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis-(HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis-(CE) and/or microchip electrophoresis-based HDA, using six heterozygous mutations, 185delAG, E1250X (3867GT), R1443G (4446CG), 5382insC, 5677insA in BRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200-300 bp, however, the effective range was 150-260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%-10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

“…The sensitivity of CE-SSCP has often been reported to be more than 90% for fragments with size of 250 bp or smaller [30,38] and the sensitivity could be increased more than 95% by using several different temperatures [32,39]. These progresses of fluorescent labeling and utilizing automated sequencer are also applied to HA [40][41][42]. Furthermore, Kozlowski et al [43] showed SSCP/HA combined analysis was achieved by CE platform and demonstrated 100% sensitivity, whereas SSCP and HA alone provided 90 and 81% sensitivity, respectively.…”

“…Furthermore, Kozlowski et al [43] showed SSCP/HA combined analysis was achieved by CE platform and demonstrated 100% sensitivity, whereas SSCP and HA alone provided 90 and 81% sensitivity, respectively. Tian et al [42] reported the upper size limit for CE-HA was 200-300 bp. Hence, both SSCP and HA remained a drawback of fragment size limitation even use automated CE platform.…”

“…Tian et al [33] have demonstrated the ability of microchip SSCP to analyze BRCA1 and BRCA2 common mutations less than 120 s. They also explored the potential of microchip HA and demonstrated high utility, but concluded fragment size upper limitation was 150-260 bp [42].…”

Development of a gel monolithic column polydimethylsiloxane microfluidic device for rapid electrophoresis separation

“…As a medium used in the microfluidic device monolithic column electrochromatograph (EC), PAA gel is a classical stationary phase for separation of proteins, enzyme and AAs [17,18]. Its application and preparation in general gel chromatography is well developed.…”

“…The group of Kato [17] had demonstrated the highly efficiency of monolithic column for electrochromatography in the separation of amino acids (AAs). Noticeably, Tian et al [18,19] have done much work in the exploring the application of gel monolithic column in life sciences for capillary or microchip electrochromatography. Based on the widely adoption of gel monolithic column in contemporary analytical science, many complicated DNA and protein have been well separated in gel monolithic column for electrochromatograohy [17][18][19].…”