We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin.

Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA / Megumi, Kobayashi; Eric, Rappaport; Andy, Blasband; Antonio, Semeraro; Maria, Sartore; Saul, Surrey; Fortina, Paolo. - In: MOLECULAR AND CELLULAR PROBES. - ISSN 0890-8508. - STAMPA. - 9:3(1995), pp. 175-182. [10.1006/mcpr.1995.0027]

Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA.

FORTINA, PAOLO
1995

Abstract

We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin.
1995
01 Pubblicazione su rivista::01a Articolo in rivista
Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA / Megumi, Kobayashi; Eric, Rappaport; Andy, Blasband; Antonio, Semeraro; Maria, Sartore; Saul, Surrey; Fortina, Paolo. - In: MOLECULAR AND CELLULAR PROBES. - ISSN 0890-8508. - STAMPA. - 9:3(1995), pp. 175-182. [10.1006/mcpr.1995.0027]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/502774
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