Random shearing as an alternative to digestion for mitochondrial DNA processing in droplet digital PCR
| Autor: | Miguel Ramirez-Gaona, Sanjay Mehta, Josué Pérez-Santiago, Andrej Vitomirov |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Biochemistry & Molecular Biology Mitochondrial DNA Mitochondrial Diseases Time Factors Sonication Biology DNA Mitochondrial Polymerase Chain Reaction Sensitivity and Specificity Article Specimen Handling 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Digestion (alchemy) Genetics Digital polymerase chain reaction Fragmentation (cell biology) Mitochondrial common deletion Molecular Biology Chromatography Enzymatic digestion Hydrolysis DNA Cell Biology Molecular biology Mitochondrial QIAshredder 030104 developmental biology Droplet digital PCR chemistry Molecular Medicine DNA fragmentation 030217 neurology & neurosurgery |
| Popis: | Droplet digital PCR (ddPCR) is a quantitative assay that requires DNA fragmentation to maximize reaction efficiency. Here, we measured the proportion of mitochondrial DNA (mtDNA) carrying the "common deletion," a rare event, to compare quantification sensitivities between alternative DNA fragmentation methods (sonication and QIAshredder spin columns) against enzymatic digestion (traditionally used). QIAshredder showed the highest sensitivity when compared to sonication, followed by digestion. Also, both sonication and QIAshredder fragmentation had shorter processing times than enzymatic digestion; therefore, QIAshredder fragmentation and sonication are alternative DNA processing methods that maximize ddPCR quantification for the detection of rare events. |
| Databáze: | OpenAIRE |
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