Sequence dependent UV damage of complete pools of oligonucleotides.

Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments.

A simple and loss-free method to remove TRIzol contaminations from minute RNA samples.

Acid guanidium phenol preparations such as TRIzol allow the reproducible isolation of high-quality total RNA from various sources. However, if applied to minimal sample sizes, the quality parameters of the isolated RNA are often low. Here we present an approach to improve the 260/280- and 230/260-nm ratios of such preparations as well as the ratio of the 18S/28S RNA. A simple extraction with 1-butanol eliminates smearing of the 28 S RNA and restores the characteristic ultraviolet (UV) spectrum of highly purified RNA. Application of the method is demonstrated for fluorescence-activated cell sorting (FACS)-sorted cells where the population of interest is often small.

Matrixlysis, an improved sample preparation method for recovery of Mycobacteria from animal tissue material.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, is the main causative agent of bovine tuberculosis in alpine regions. Bacterial culture is the gold standard in bovine tuberculosis diagnostic but takes up to twelve weeks. This increases the time and costs for stocks affected with bovine tuberculosis. Hence this study focused on the implementation of a fast and precise mycobacterial detection method and compared it with currently used methods. Matrix lysis is a chemical lysis using high concentrations of urea to solubilize bovine and red deer tissue and was used to detect even smallest amounts or non-visible lesions of mycobacteria. A total of 64 samples collected from 44 animals (37 red deer and 7 cattle) were tested by Matrix lysis. Forty-three of these samples were used for Mycobacterium tuberculosis complex detection by quantitative PCR and other 21 for subtyping the genetically different variants of M. caprae. Furthermore, three Matrix lysis samples were used for Next Generation Sequencing. Our results confirm that Matrix lysis is a fast and precise method for detecting Mycobacterium tuberculosis complex in native tissue samples. However, at the moment it reaches its limits when the samples were analyzed by Next Generation Sequencing and RD4 subtyping.