Maximizing confidence in a negative result: Quantitative sample adequacy control.

Quantitative PCR (qPCR) is a leading screening tool, permitting rapid detection of pathogens and the maintenance of effective infection control programs. Unfortunately, qPCR assays frequently do not incorporate Sample Adequacy Control (SAC). A SAC controls for the quantity, quality and adequacy of the specimen. Without SAC, the confidence in a negative result remains questionable and the efficacy of screening is compromised. Ultimately, the exclusion of SAC from qPCR may result in false negative results. One should consider SAC to be an integral critical type of laboratory control; addressing diverse analytical problems, such as sample adequacy, sample processing and assay inhibition. Following distribution of cycle threshold values (Cq) of Influenza A positive results and Cq values of SAC, obtained from nasopharyngeal swabs, we showed that the confidence in a negative result cannot be guaranteed in the presence of a weak positive SAC signal (late Cq values). Herein, we explain why widespread inclusion of sample adequacy control in routine screening is blocked. A protocol and methods for SAC threshold establishment are offered.

Genome-wide mapping of DNA strand breaks.

Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

A mutation in yeast Tel1p that causes differential effects on the DNA damage checkpoint and telomere maintenance.

ATM/ATR homologs are the central elements of genome surveillance mechanisms in many organisms, including yeasts, flies, and mammals. In Saccharomyces cerevisiae, most checkpoint responses depend on the ATR ortholog Mec1p. The yeast ATM ortholog, Tel1p, so far has been implicated in a specific DNA damage checkpoint during S-phase as well as in telomere homeostasis. In particular, yeast cells lacking only Tel1p harbor short but stable telomeres, while cells lacking both Tel1p and Mec1p are unable to maintain telomeric repeats and senesce. Here, we present the characterization of a new mutation in the TEL1-gene, called tel1-11, which was isolated by virtue of a synthetic lethal interaction at 37 degrees C with a previously described mec1-ts mutation. Interestingly, telomere and checkpoint functions are differentially affected by the mutant protein Tel1-11p. The Tel1p-dependent checkpoint response is undetectable in cells containing Tel1-11p and incubated at 37 degrees C, but basic telomere function is maintained. Further, when the same cells are incubated at 26 degrees C, Tel1-11p confers full proficiency for all telomere functions analyzed, whereas the function for DNA-damage checkpoint activation is clearly affected. The results thus strongly suggest that the different cellular pathways affected by Tel1p do not require the same level of Tel1p activity to be fully functional.