Temperature-Assisted Cyclic Hybridization (TACH): An Improved Method for Supercoiled DNA Hybridization
| Autor: | Oscar E. Simonson, Joana R Viola, Pedro Moreno, C. I. Edvard Smith, Karin E. Lundin, Iulian I. Oprea |
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| Rok vydání: | 2010 |
| Předmět: |
Peptide Nucleic Acids
Gene Expression Bioengineering In situ hybridization Biology Sensitivity and Specificity Applied Microbiology and Biotechnology Biochemistry Mice chemistry.chemical_compound Nucleic acid thermodynamics Molecular beacon medicine Animals Locked nucleic acid Molecular Biology Electrophoresis Agar Gel Analysis of Variance Peptide nucleic acid medicine.diagnostic_test DNA Superhelical Hybridization probe Temperature Nucleic Acid Hybridization Hydrogen-Ion Concentration chemistry Nucleic acid Nucleic Acid Conformation Plasmids Biotechnology Fluorescence in situ hybridization |
| Zdroj: | Molecular Biotechnology. 45:171-179 |
| ISSN: | 1559-0305 1073-6085 |
| Popis: | Accurate hybridization is dependent on the ratio between sequence-specific and unspecific binding. Dissociation of unspecifically bound, while maintaining specifically hybridized, nucleic acids are key steps to obtain a well-defined complex. We have developed a new method, temperature-assisted, cyclic hybridization (TACH), which increases cognate binding at the expense of unspecific hybridization. The method was used for optimizing binding of peptide nucleic acid (PNA) to supercoiled plasmids and has several advantages over previous methods: (1) it reduces the required amount of bis-PNA by three- to fourfold; (2) it results in less unspecific binding; (3) it extends cooperative hybridization, from 3 bp to 5 bp between two adjacent binding sites; and (4) it decreases the aggregation of bis-PNA. This method might be extended to other forms of hybridizations including the use of additional nucleic acids analogs, such as locked nucleic acid (LNA) and, also, to other areas where PNAs are used such as fluorescence in situ hybridization (FISH), microarrays, or in vivo plasmid delivery. |
| Databáze: | OpenAIRE |
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