| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260.
2
Genetics and Molecular Biology Branch, National Human
Genome Research Institute, National Institutes of Health, Bethesda, MD
20892.
3
Department of Radiation Oncology, Long Island Jewish
Medical Center, Albert Einstein College of Medicine, New Hyde Park, NY
11040.
4
Department of Chemistry, University of Virginia,
Charlottesville, VA 22901.
5
Department of Pathology, University of Virginia Medical
Center, Charlottesville, VA 22908.
a Address correspondence to this author at: Department of Chemistry, University of Virginia, McCormick Rd., Charlottesville, VA 22901. Fax 412-243-8852; e-mail jpl5e{at}virginia.edu.
Background: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification.
Methods: DNA samples heterozygous for four mutations (185delAG,
5382insC, 3867G
T, and 6174delT) in BRCA1 and
BRCA2, and homozygous for one mutation (5382insC)
in BRCA1 and two mutations (16delAA and 822delG) in
PTEN were chosen as the model system to evaluate the
capillary and microchip electrophoresis methods. To detect each
mutation, three primers, of which one was labeled with the fluorescent
dye 6-carboxyfluorescein and one was the allele-specific primer
(mutation-specific primer), were used to amplify the DNA fragments in
the range of 130–320 bp. AS-PCR was combined with heteroduplex (HD)
analysis, where the DNA fragments obtained by AS-PCR were analyzed with
the conditions developed for CE-based HD analysis (using a
fluorocarbon-coated capillary and hydroxyethylcellulose). The CE
conditions were transferred into the microchip electrophoresis format.
Results: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10–25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less.
Conclusions: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  J. Chowdhury, G. V. Kagiala, S. Pushpakom, J. Lauzon, A. Makin, A. Atrazhev, A. Stickel, W. G. Newman, C. J. Backhouse, and L. M. Pilarski Microfluidic Platform for Single Nucleotide Polymorphism Genotyping of the Thiopurine S-Methyltransferase Gene to Evaluate Risk for Adverse Drug Events J. Mol. Diagn., September 1, 2007; 9(4): 521 - 529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Hung, C.-P. Chen, S.-P. Lin, S.-C. Chien, C.-N. Lee, W.-F. Cheng, W.-S. Hsieh, M. S. Liu, Y.-N. Su, and W.-L. Lin Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader-Willi Syndrome and Duchenne Muscular Dystrophy Clin. Chem., December 1, 2006; 52(12): 2203 - 2210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F.Y. Li and L. J. Kricka Clinical Analysis by Microchip Capillary Electrophoresis Clin. Chem., January 1, 2006; 52(1): 37 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Esteban-Cardenosa, M. Duran, M. Infante, E. Velasco, and C. Miner High-Throughput Mutation Detection Method to Scan BRCA1 and BRCA2 Based on Heteroduplex Analysis by Capillary Array Electrophoresis Clin. Chem., February 1, 2004; 50(2): 313 - 320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Paegel, C. A. Emrich, G. J. Wedemayer, J. R. Scherer, and R. A. Mathies High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor PNAS, January 22, 2002; 99(2): 574 - 579. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
