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Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays

BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. T...

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Detalles Bibliográficos
Autores principales: Poulsen, Lena, Søe, Martin Jensen, Møller, Lisbeth Birk, Dufva, Martin
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062541/
https://www.ncbi.nlm.nih.gov/pubmed/21445337
http://dx.doi.org/10.1371/journal.pone.0014777
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author Poulsen, Lena
Søe, Martin Jensen
Møller, Lisbeth Birk
Dufva, Martin
author_facet Poulsen, Lena
Søe, Martin Jensen
Møller, Lisbeth Birk
Dufva, Martin
author_sort Poulsen, Lena
collection PubMed
description BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20–75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.
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spelling pubmed-30625412011-03-28 Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays Poulsen, Lena Søe, Martin Jensen Møller, Lisbeth Birk Dufva, Martin PLoS One Research Article BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20–75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios. Public Library of Science 2011-03-22 /pmc/articles/PMC3062541/ /pubmed/21445337 http://dx.doi.org/10.1371/journal.pone.0014777 Text en Poulsen et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Poulsen, Lena
Søe, Martin Jensen
Møller, Lisbeth Birk
Dufva, Martin
Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title_full Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title_fullStr Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title_full_unstemmed Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title_short Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays
title_sort investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062541/
https://www.ncbi.nlm.nih.gov/pubmed/21445337
http://dx.doi.org/10.1371/journal.pone.0014777
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