An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma
Sarah A. WareNikita DesaiMabel LopezDaniel LeachYingze ZhangLuca GiordanoMehdi NouraieMartin PicardBrett A. Kaufman
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Abstract:
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.Keywords:
Nuclear DNA
Magnetic bead
Nuclear DNA
Forensic Genetics
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To explore an efficient,stable,low-cost DNA extraction method from single nematode for the support of molecular identification of nematodes,using six species of plant-parasitic nematodes as experimental materials, four DNA extraction methods,namely,direct lysis method,freeze-thaw lysis method,cutting lysis method,freeze-thaw cutting lysis method were designed and compared in order to check their efficiency of DNA extraction from single nematode. The results showed that cutting lysis method and freeze-thaw cutting lysis method were the most efficient and stable methods for DNA extraction of single nematode,then the freeze-thaw lysis method,and the direct lysis method was the worst one. Total DNA with high-quality from a single nematode could be gained efficiently and stably through two methods,namely,cutting lysis method and freeze-thaw cutting lysis method. And the extracted DNA meeted requirement of the following PCR amplification.
Alkaline lysis
Lysis buffer
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Analysis of the mitochondrial DNA (mtDNA) is an important part in the diagnosis of mitochondrial disorders. Besides point mutations and deletions in the mitochondrial genome a reduction in the amount of mtDNA molecules (mtDNA depletion) can also be the reason for mitochondrial defects. The DNA stability in clinical samples is essential for proper performance and interpretation of DNA based diagnosis. The stability of mtDNA was compared with that of nuclear DNA under poor handling and storage conditions. Fresh and thawed muscle tissue specimens were kept at different temperatures for a certain period of time before DNA isolation. Quantitative Southern blot analysis revealed a time-dependent decrease in the amount of mtDNA compared with nuclear DNA in thawed tissue specimens. Therefore, the current study demonstrates that proper specimen storage is a critical issue in quantitative mtDNA analysis and that poor handling and storage of tissue may mimic a severe mtDNA depletion.
Nuclear DNA
Southern blot
Mitochondrial disease
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Nuclear gene
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Nuclear DNA
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Nuclear DNA
Nuclear gene
Human mitochondrial genetics
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To isolate DNA from liquid blood and forensic samples using magnetic bead-based extraction system.
Magnetic bead
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Isolation
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A bstract : Since 1988 several research groups have reported greater levels of oxidative damage in mitochondrial DNA than in nuclear DNA, while others have suggested that the greater damage in mtDNA might be due to artifactual oxidation. The popular theory that mtDNA is more heavily damaged in vivo than nDNA does not stand on firm ground. Using an improved GC‐MS method and pure mtDNA, our analyses revealed that the damage level in mtDNA is not higher, and may be somewhat lower, than that in nDNA.
Nuclear DNA
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Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that ΦX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference for quantifying mtDNA copy number.
Nuclear DNA
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