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The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.

ABSTRACT: The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA repair, and describe methodological changes implemented in recent years. We present QPCR assay primers and reaction conditions for five species not previously described in a methods article: Caenorhabditis elegans, Fundulus heteroclitus, Danio rerio, Drosophila melanogaster, and adenovirus. Finally, we illustrate the use of the assay by measuring repair of ultraviolet C radiation-induced DNA damage in the nuclear but not mitochondrial genomes of a zebrafish cell culture.

SUBMITTER: Hunter SE

PROVIDER: S-EPMC2912960 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.

Hunter Senyene E SE Jung Dawoon D Di Giulio Richard T RT Meyer Joel N JN

Methods (San Diego, Calif.) 20100201 4

The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA ...[more]

PMID: 20123023

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Project description:Rationale: Loss of DNA damage repair (DDR) in the tumor is an established hallmark of sensitivity to DNA damaging agents such as chemotherapy. However, there has been scant investigation into gain-of-function alterations of DDR genes in cancer. This study aims to investigate to what extent copy number amplification of DDR genes occurs in cancer, and what are their impacts on tumor genome instability, patient prognosis and therapy outcome. Methods: Retrospective analysis was performed on the clinical, genomics, and pharmacogenomics data from 10,489 tumors, matched peripheral blood samples, and 1,005 cancer cell lines. The key discoveries were verified by an independent patient cohort and experimental validations. Results: This study revealed that 13 of the 80 core DDR genes were significantly amplified and overexpressed across the pan-cancer scale. Tumors harboring DDR gene amplification exhibited decreased global mutation load and mechanism-specific mutation signature scores, suggesting an increased DDR proficiency in the DDR amplified tumors. Clinically, patients with DDR gene amplification showed poor prognosis in multiple cancer types. The most frequent Nibrin (NBN) gene amplification in ovarian cancer tumors was observed in 15 out of 31 independent ovarian cancer patients. NBN overexpression in breast and ovarian cancer cells leads to BRCA1-dependent olaparib resistance by promoting the phosphorylation of ATM-S1981 and homology-dependent recombination efficiency. Finally, integration of the cancer pharmacogenomics database of 37 genome-instability targeting drugs across 505 cancer cell lines revealed significant correlations between DDR gene copy number amplification and DDR drug resistance, suggesting candidate targets for increasing patient treatment response. Principal Conclusions: DDR gene amplification can lead to chemotherapy resistance and poor overall survival by augmenting DDR. These amplified DDR genes may serve as actionable clinical biomarkers for cancer management.

Project description:BACKGROUND: Some diseases, like tumors, can be related to chromosomal aberrations, leading to changes of DNA copy number. The copy number of an aberrant genome can be represented as a piecewise constant function, since it can exhibit regions of deletions or gains. Instead, in a healthy cell the copy number is two because we inherit one copy of each chromosome from each our parents. Bayesian Piecewise Constant Regression (BPCR) is a Bayesian regression method for data that are noisy observations of a piecewise constant function. The method estimates the unknown segment number, the endpoints of the segments and the value of the segment levels of the underlying piecewise constant function. The Bayesian Regression Curve (BRC) estimates the same data with a smoothing curve. However, in the original formulation, some estimators failed to properly determine the corresponding parameters. For example, the boundary estimator did not take into account the dependency among the boundaries and succeeded in estimating more than one breakpoint at the same position, losing segments. RESULTS: We derived an improved version of the BPCR (called mBPCR) and BRC, changing the segment number estimator and the boundary estimator to enhance the fitting procedure. We also proposed an alternative estimator of the variance of the segment levels, which is useful in case of data with high noise. Using artificial data, we compared the original and the modified version of BPCR and BRC with other regression methods, showing that our improved version of BPCR generally outperformed all the others. Similar results were also observed on real data. CONCLUSION: We propose an improved method for DNA copy number estimation, mBPCR, which performed very well compared to previously published algorithms. In particular, mBPCR was more powerful in the detection of the true position of the breakpoints and of small aberrations in very noisy data. Hence, from a biological point of view, our method can be very useful, for example, to find targets of genomic aberrations in clinical cancer samples.

Dataset Information

The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.

Publications

The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.

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