Selection of oligonucleotide probes and experimental conditions for multiplex hybridization experiments |
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| Institution: | 1. Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI, USA;2. Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA;3. Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI, USA;4. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA;5. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA;6. Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA;7. Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA;1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China;2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China;3. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia;4. Department of Microbiology, Guiyang Medical University, Guiyang, China;1. Cl 18 # 122-135, Departamento de Mercadeo y Negocios Internacionales, Universidad Icesi, Cali, Colombia;2. Cl 18 # 122-135, Departamento de Tecnologías de Información y Comunicaciones, Universidad Icesi, Cali, Colombia |
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| Abstract: | Different DNA probes hybridize under different conditions. I examine the constraints of the design of oligonucleotide probes that are meant to hybridize to different unique sites in human genomic DNA under a single set of hybridization conditions as a parallel array. In 522 kb of human genomic DNA, 75% of 12-base and 89% of 22-base are unique, as opposed to 90% and 100% as expected of unstructured DNA, and this is not due solely to repetitive elements in the DNA. Hybridization in TMAC to reduce A + T content effects on melting temperature allows only 90% of unique targets to be hybridized under one set of conditions if a 2°C difference between matched and mismatched sequences is required. Standard hybridization conditions allow no more than 60% of unique probes to be used together. This suggests that probe, hybridization conditions, and instrument design for multiple-probe hybridization applications will be harder than previously suggested. |
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