Pitfalls in the Denaturing High-Performance Liquid Chromatography Analysis of Mitochondrial DNA Mutation
| Autor: | Kok Seong Lim, Robert K. Naviaux, Richard H. Haas, Scott T. Wong |
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| Rok vydání: | 2008 |
| Předmět: |
Mitochondrial DNA
RNA Transfer Leu DNA Mutational Analysis Molecular Sequence Data Mutant Heteroduplex Analysis Biology Nucleic Acid Denaturation DNA Mitochondrial DNA sequencing Pathology and Forensic Medicine Denaturing high performance liquid chromatography chemistry.chemical_compound Humans Chromatography High Pressure Liquid Base Sequence Temperature Molecular biology Heteroplasmy chemistry Mutation Molecular Medicine Female DNA Regular Articles Heteroduplex |
| Zdroj: | The Journal of Molecular Diagnostics. 10:102-108 |
| ISSN: | 1525-1578 |
| Popis: | Denaturing high-performance liquid chromatography (DHPLC) purification of heteroduplexes has been reported as a method to increase sensitivity of the detection of low-level heteroplasmy by DNA sequencing, and DHPLC profiling has been suggested as a method to allow the correlation of a characteristic chromatographic profile with a specific sequence alteration. Herein we report pitfalls associated with the use of DHPLC for these purposes. We show that the purified heteroduplex fraction does not contain a 50:50 mix of wild-type and mutant DNA in DNA samples containing low-level mutations, and that with a commonly used protocol, DNA sequencing gave false negative results at the 1% mutation level, potentially leading to misdiagnosis. We improved the protocol to detect low levels of mutations and evaluated the sensitivity of DNA sequencing in the detection of mutation in these fractions. We also studied the DHPLC profiles of several mutations in the tRNALeu(UUR) region of mitochondrial DNA and found a characteristic profile in only one of five mutants tested, whereas four other mutants showed identical chromatographic profiles. |
| Databáze: | OpenAIRE |
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