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| November 2000, Volume 8, Number 11, Pages 884-894 |
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| Article |
| Simple two-color array-based approach for mutation detection |
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| Paolo Fortina1, Kathleen Delgrosso2, Taku Sakazume1, Rosa Santacroce1, Stephane Moutereau1, Hung-Ju Su1,3, David Graves3, Steven McKenzie2,4 and Saul Surrey2,4 |
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1Department of Pediatrics, University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, PA
2duPont Hospital for Children, Wilmington, DE
3Department of Chemical Engineering University of Pennsylvania School of Engineering and Applied Science, Philadelphia, PA
4Department of Pediatrics, Jefferson College of Medicine, Philadelphia, PA
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Correspondence to: Paolo Fortina MD, The Children's Hospital of Philadelphia, 310-C Abramson Pediatric Research Center, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, USA. Tel: +1 215 590 3318; Fax: +1 215 590 3660; E-mail: fortina@mail.med.upenn.edu
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| Abstract |
| The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis European Journal of Human Genetics (2000) 8, 884-894. |
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| Keywords |
| array-bound single nucleotide primer extension; minisequencing; fluorescent ddNTPs; mutation detection |
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| Received 8 May 2000; revised 14 July 2000; accepted 18 July 2000 |
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| November 2000, Volume 8, Number 11, Pages 884-894 |
| Table of contents Previous Abstract Next Article PDF |
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