Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis

Ulfelder, Kathi J.; Schwartz, H. E.; Hall, Jeff; Sunzeri, Franklin J.

doi:10.1016/0003-2697(92)90463-h

Cited by 112 publications

(24 citation statements)

References 16 publications

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“…While PCR product analysis by CE using entangled polymer solutions is relatively immune to the high salt concentrations of PCR mixtures (Ulfelder et al 1992;Guttman and Schwartz 1995;Pancholi et al 1997), work by several groups (Woolley and Mathies 1994;Woolley et al 1997;Munro et al 1999;Shi et al 1999) indicated that salt can degrade separations on microchips. The effect of the sample salt concentration on CE-based HDA was tested using a com- Figure 1 Effect of glycerol on capillary electrophoresis-based heteroduplex analysis of 5677insA mutant.…”

Section: Effect Of Salt On Ce-based Heteroduplex Analysismentioning

confidence: 99%

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

Tian¹,

Brody²,

Landers³

2000

Genome Res.

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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.

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Section: Effect Of Salt On Ce-based Heteroduplex Analysismentioning

confidence: 99%

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

Tian¹,

Brody²,

Landers³

2000

Genome Res.

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“…However, an increase in the UV absorbance (about 5 fold for 872 bp) and a decrease in the migration time of DNA fragments were observed upon the addition. Therefore, dextran sulfate probably changes the structure and charge of DNA during electrophoresis, similar to the effect of an intercalation dye reported by Schwartz et al 6 Further, the optimal conditions of the capillary length and voltage were determined as described in method. The separation of a 100-base ladder of a DNA marker obtained under these optimal conditions is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Resolution in the Capillary Electrophoretic Separation of Double-Stranded DNA Using Dextran Sulfate and Its Application to the Analysis of PCR Products

et al. 1997

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“…Various adaptations of common gel electrophoresis methods and applications previously developed on home-built CE instruments have thus become routine on commercial systems. The most popular CE applications, other than sequencing or microsatellite genotyping, utilize fragment sizing by RFLP [6], amplified fragment length polymorphism (AFLP) [7], or following multiplexed ligation [8,9] and allele separation by SSCP [10] or heteroduplex analysis [11]. A special group of methods employs separation due to partial DNA melting in CE-adapted versions of denaturing gradient gel electrophoresis techniques [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Parallel optimization and genotyping of multiple single‐nucleotide polymorphism markers by sample pooling approach using cycling‐gradient CE with multiple injections

Minárik¹,

Benešová²,

Fantova³

et al. 2006

Electrophoresis

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Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.

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Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis

Cited by 112 publications

References 16 publications

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

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

Enhanced Resolution in the Capillary Electrophoretic Separation of Double-Stranded DNA Using Dextran Sulfate and Its Application to the Analysis of PCR Products

Parallel optimization and genotyping of multiple single‐nucleotide polymorphism markers by sample pooling approach using cycling‐gradient CE with multiple injections

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