Nicole R. Phillips

Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: A multiplex real-time PCR assay

Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimers disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually.

Mitochondrial DNA deletions in Alzheimer's brains: a review.

Mitochondrial dysfunction and increased oxidative stress have been associated with normal aging and are possibly implicated in the etiology of late‐onset Alzheimers disease (AD). DNA deletions, as well as other alterations, can result from oxidative damage to nucleic acids. Many studies during the past two decades have investigated the incidence of mitochondrial DNA deletions in postmortem brain tissues of late‐onset AD patients compared with age‐matched normal control subjects. Published studies are not entirely concordant, but their differences might shed light on the heterogeneity of AD itself. Our understanding of the role that mitochondrial DNA deletions play in disease progression may provide valuable information that could someday lead to a treatment.

Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees.

Aim To perform a genetic characterization of 7 skeletons from medieval age found in a burial site in the Aragonese Pyrenees. Methods Allele frequencies of autosomal short tandem repeats (STR) loci were determined by 3 different STR systems. Mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were determined by sequencing of the hypervariable segment 1 of mtDNA and typing of phylogenetic Y chromosome single nucleotide polymorphisms (Y-SNP) markers, respectively. Possible familial relationships were also investigated. Results Complete or partial STR profiles were obtained in 3 of the 7 samples. Mitochondrial DNA haplogroup was determined in 6 samples, with 5 of them corresponding to the haplogroup H and 1 to the haplogroup U5a. Y-chromosome haplogroup was determined in 2 samples, corresponding to the haplogroup R. In one of them, the sub-branch R1b1b2 was determined. mtDNA sequences indicated that some of the individuals could be maternally related, while STR profiles indicated no direct family relationships. Conclusions Despite the antiquity of the samples and great difficulty that genetic analyses entail, the combined use of autosomal STR markers, Y-chromosome informative SNPs, and mtDNA sequences allowed us to genotype a group of skeletons from the medieval age.

Optimization of primer specific filter metrics for the assessment of mitochondrial DNA sequence data

Filter metrics are used as a quick assessment of sequence trace files in order to sort data into different categories (i.e. high quality, review, and low quality) without human intervention. The filter metrics consist of two numerical parameters for sequence quality assessment: trace score (TS) and contiguous read length (CRL). Primer-specific settings for the TS and CRL were established using a calibration dataset of 2817 traces and validated using a concordance dataset of 5617 traces. Prior to optimization, 57% of the traces required manual review before import into a sequence analysis program, whereas after optimization only 28% of the traces required manual review. After optimization of primer-specific filter metrics for mitochondrial DNA sequence data, an overall reduction of review of trace files translates into increased throughput of data analysis and decreased time required for manual review.

Etiology of type 2 diabetes and Alzheimer's disease: Exploring the mitochondria

Type 2 diabetes is a significant risk factor for developing Alzheimers disease later in life, and particular populations have a disproportionate risk because of the high prevalence of type 2 diabetes. There are many overlapping pathologies, and teasing out the primary root cause, if one indeed exists, is very difficult. Here, we review (1) the key facets of mitochondrial biology that are relevant to the two conditions, and (2) the role that mitochondrial dysfunction plays in the shared pathophysiology. We posit that mitochondrial dysfunction lies at the root of the affected processes rather than alongside them as a co-pathology.

Mitochondrial Genome Interrogation for Forensic Casework and Research Studies

This unit describes methods used in the analysis of mitochondrial DNA (mtDNA) for forensic and research applications. UNIT describes procedures specifically for forensic casework where the DNA from evidentiary material is often degraded or inhibited. In this unit, protocols are described for quantification of mtDNA before amplification; amplification of the entire control region from high‐quality samples as well as procedures for interrogating the whole mitochondrial genome (mtGenome); quantification of mtDNA post‐amplification; and, post‐PCR cleanup and sequencing. The protocols for amplification were developed for high‐throughput databasing applications for forensic DNA testing such as reference samples and population studies. However, these same protocols can be applied to biomedical research such as age‐related disease and health disparities research. Curr. Protoc. Hum. Genet. 81:14.9.1‐14.9.23.