A single-tube multiplex qPCR assay for mitochondrial DNA (mtDNA) copy number assessment

Objective Detection of mtDNA copy number is required for diagnosis of mtDNA depletion. Multiplex quantification of mtDNA in blood samples was claimed via normalizing to a nuclear single copy gene using qPCR. This is not possible in high mtDNA samples due to template abundance. Multiplex qPCR assays cannot be normalized to single copy sequences of the nuclear genome. Methods mtDNA quantification was tested normalizing to a single copy nuclear gene via singleplex and multiplex reactions. Failure in normalization directed to design and test targeting multi-copy 18S rDNA gene with success. mtDNA quantification was standardized both in separate and multiplexed single-tube reactions based on molecular beacon technology. Results mtDNA copy number assessment cannot be normalized to a single copy sequence in high-copy-number tissues. However, normalizing mtDNA to the nuclear 18S rDNA multiple copy sequence is amenable to be standardized in single tube. When compared, multiplexing exhibited higher resolution power for quantification of mtDNA in various samples from the most abundant to the scant ones. Conclusion We describe a multiplex assay that can be translated as a standard technique for single-tube quantification of mtDNA copy number. Our findings show higher accuracy and reproducibility over canonical approach, reducing cost and error rate.

Nonequilibrium correlations in minimal dynamical models of polymer copying

Living systems produce “persistent” copies of information-carrying polymers, in which template and copy sequences remain correlated after physically decoupling. We identify a general measure of the thermodynamic efficiency with which these nonequilibrium states are created and analyze the accuracy and efficiency of a family of dynamical models that produce persistent copies. For the weakest chemical driving, when polymer growth occurs in equilibrium, both the copy accuracy and, more surprisingly, the efficiency vanish. At higher driving strengths, accuracy and efficiency both increase, with efficiency showing one or more peaks at moderate driving. Correlations generated within the copy sequence, as well as between template and copy, store additional free energy in the copied polymer and limit the single-site accuracy for a given chemical work input. Our results provide insight into the design of natural self-replicating systems and can aid the design of synthetic replicators.

Unsupervized Quantification of Demographic Structure for Single-copy Alignments

Single-copy sequence alignments have been a valuable source of information for genetic studies; their lack of recombination makes phylogenetic analyses tractable [1]. Specifically, mitochondrial DNA will continue to play an important role in genetic studies due to its high mutation rate and high copy per cell count of the molecule [2]. In this paper we develop a new method for the analysis of single-copy sequence data that simultaneously considers the relationships between sequenced individuals and positions of interest in the genome. We then show that tests for relationships between genetic information and qualitative and quantitative characteristics can be calculated. We motivate the use of our method with examples from empirical data.

Isolation and characterisation of repeated DNA sequences from Erianthus spp. (Saccharinae: Andropogoneae)

Four anonymous noncoding sequences were isolated from Erianthus arundinaceus. The four sequences were selected because they were specific to the genusErianthus section Ripidium, relative to Saccharum spp. These sequences, designated Eracsi 294, 228, 153, and 34, showed various degrees of repetitiveness and different patterns of distribution. Eracsi 34 and 153 were low- and medium-copy repeated sequences, respectively, and appeared to be present at discrete locations in the Erianthus genome. By contrast, Eracsi 294, also a low-copy sequence, appeared to be more dispersed in location, with some tandem arrays identified. Eracsi 228 was highly repeated and dispersed. The location of Eracsi 228 was more precisely determined by FISH and was found to be distributed along the length of, but not at the telomeres of, most chromosomes in two Erianthus species. The distribution of the four sequences was investigated in a sample of 65 Erianthus (representing 9 species) and 14 Saccharum (2 species) accessions. The usefulness of these sequences for phylogenetic and genome organisation studies in sugarcane and for assessing the genetic structure of Saccharum x Erianthus intergeneric hybrids is discussed.Key words: Erianthus, FISH, repetitive sequences, Saccharum, sugarcane.