European Journal of Human Genetics

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bold italic beta-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays

Coline H M van Moorsel, Erwin E van Wijngaarden, Ivo F A C Fokkema, Johan T den Dunnen, Dirk Roos, Rob van Zwieten, Piero C Giordano and Cornelis L Harteveld

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Figure 1.

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The amplified PCR product is used as a template for the SBE reaction, which is performed in a thermal cycler with a set of 18 mutation-specific primers. Each primer is elongated at the 3' site with a specific ddNTP, complementary to the wild-type or the mutation. By splitting the SBE reaction into two separate reactions including ddATPCy5/ddUTPCy3/ddGTP/ddCTP and ddGTPCy5/ddCTPCy3/ddATP/ddTTP, all possible combinations can be detected. Each mutation-specific primer contains a unique 20-mer tag at the 5'site that will hybridize to a spotted complementary sequence on the generic array. A fluorescent signal was detected through scanning and a CCD camera for glass and flow-through microarrays, respectively. Open asterisk, Cy3; closed asterisk, Cy5.

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