Rapid Genotyping of Common MeCP2 Mutations with an Electronic DNA Microchip Using Serial Differential Hybridization William A. Thistlethwaite, Linda.

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Rapid Genotyping of Common MeCP2 Mutations with an Electronic DNA Microchip Using Serial Differential Hybridization William A. Thistlethwaite, Linda M. Moses, Kristen C. Hoffbuhr, Joseph M. Devaney, Eric P. Hoffman The Journal of Molecular Diagnostics Volume 5, Issue 2, Pages 121-126 (May 2003) DOI: 10.1016/S1525-1578(10)60461-8 Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of the Nanogen differential hybridization process. A: Attachment of a single set of PCR amplimers on one pad of the NanoChip. B: Fluorescent oligonucleotide probes specific to the wild-type (labeled with Cy-3) and mutant (labeled with Cy-5) base are added to the PCR product-loaded pad. In this case, one probe is a match, the other is mismatched for the base in question. In addition, a 27 to 33-mer stabilizer oligo is added to the pad simultaneously to bind to the complementary area directly 5′ of the mutation. C: Thermal stringency then strips an improperly binding probe while leaving the correct one bound. D: Multiplex binding of two amplimers to the same pad of the NanoChip. The subsequent probing is done serially with distinct probes and stabilizers for one amplimer, followed by thermal stringency and washing, and then the next amplimer is tested. The Journal of Molecular Diagnostics 2003 5, 121-126DOI: (10.1016/S1525-1578(10)60461-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Nanogen Reader results for four samples that were heterozygous for MeCP2 mutations and six samples that were homozygous wild-type. After loading onto NanoChips, samples were read by the Nanogen Reader. Arbitrary fluorescence units were measured for both wild-type (Cy-3) and mutant (Cy-5) signals in all samples. The Journal of Molecular Diagnostics 2003 5, 121-126DOI: (10.1016/S1525-1578(10)60461-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Nanogen system results for 368 tested samples. After detection of fluorescence, wild-type (Cy-3) and mutant (Cy-5) signals were normalized by a control heterozygous sample. The Nanogen software then used the ratio of the normalized data, shown above, to genotype the sample. The generated results were in agreement with those from previous DHPLC and sequencing. The Journal of Molecular Diagnostics 2003 5, 121-126DOI: (10.1016/S1525-1578(10)60461-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Nanogen system results for testing of seven samples. For the samples, each point mutation was probed serially. After detection of fluorescence, calls were made in the same manner as non-multiplexed samples and probes were subsequently stripped, readying the samples for the next test. The generated results were in agreement with those from previous DHPLC and sequencing. The Journal of Molecular Diagnostics 2003 5, 121-126DOI: (10.1016/S1525-1578(10)60461-8) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions