Saliva-derived DNA performs well in large-scale, high-density single-nucleotide polymorphism microarray studies

As of June 2009, 361 genome-wide association studies (GWAS) had been referenced by the HuGE database. GWAS require DNA from many thousands of individuals, relying on suitable DNA collections. We recently performed a multiple sclerosis (MS) GWAS where a substantial component of the cases (24%) had DNA derived from saliva. Genotyping was done on the Illumina genotyping platform using the Infinium Hap370CNV DUO microarray. Additionally, we genotyped 10 individuals in duplicate using both saliva- and blood-derived DNA. The performance of blood- versus saliva-derived DNA was compared using genotyping call rate, which reflects both the quantity and quality of genotyping per sample and the “GCScore,” an Illumina genotyping quality score, which is a measure of DNA quality. We also compared genotype calls and GCScores for the 10 sample pairs. Call rates were assessed for each sample individually. For the GWAS samples, we compared data according to source of DNA and center of origin. We observed high concordance in genotyping quality and quantity between the paired samples and minimal loss of quality and quantity of DNA in the saliva samples in the large GWAS sample, with the blood samples showing greater variation between centers of origin. This large data set highlights the usefulness of saliva DNA for genotyping, especially in high-density single-nucleotide polymorphism microarray studies such as GWAS.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk and epigenetics also plays a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-Wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485 000 CpG sites using the Illumina Infinium Human Methylation450 Bead Chip. After adjusting for age and post-mortem interval (PMI), 4 641 probes corresponding to 2 929 unique genes were found to be differentially methylated. Of those genes, 1 291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10 which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27 000 CpG sites were analysed. Unsupervised clustering analysis of the top 3 000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared to controls (p = 1.74x10-4). The first cluster was composed of 88% of patients with schizophrenia and only 12% controls while the second cluster was composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

Ceramic membranes for separation of proteins and DNA by in situ growth of alumina nanofibres with a nanomesh of metal oxide nanofibres

Ceramic membranes were fabricated by in situ synthesis of alumina nanofibres in the pores of an alumina support as a separation layer, and exhibited a high permeation selectivity for bovine serum albumin relative to bovine hemoglobin (over 60 times) and can effectively retain DNA molecules at high fluxes.

Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence analysis extracted the important optimum constant wavelength differences, Δλ, and showed that for the TC–Al3+–DNA, TC–Al3+ and MB dye systems, the associated Δλ values were different (Δλ = 80, 75 and 30 nm, respectively). Subsequent PARAFAC analysis demonstrated the extraction of the equilibrium concentration profiles for the TC–Al3+, TC–Al3+–DNA and MB probe systems. This information is unobtainable by conventional means of data interpretation. The results indicated that the MB dye interacted with the TC–Al3+–DNA surface complex, presumably via a reaction intermediate, TC–Al3+–DNA–MB, leading to the displacement of the TC–Al3+ by the incoming MB dye probe.

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4R24C/R24C/Tyr-NrasQ61K mice following neonatal UVR

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4R24C/R24C/NrasQ61K mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.

Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, WST-1, and MTT, which were originally developed to determine cell toxicity, are being used to assess cell numbers. Additionally, proliferative activity can be determined by quantification of DNA content using fluorophores, such as CyQuant and PicoGreen. Referring to data published in high ranking cancer journals, 945 publications applied these assays over the past 14 years to examine the proliferative behaviour of diverse cell types. Within this study, mainly metabolic assays were used to quantify changes in cell growth yet these assays may not accurately reflect cellular proliferation rates due to a miscorrelation of metabolic activity and cell number. Testing this hypothesis, we compared metabolic activity of different cell types, human cancer cells and primary cells, over a time period of 4 days using AlamarBlue and fluorometric assays CyQuant and PicoGreen to determine their DNA content. Our results show certain discrepancies in terms of over-estimation of cell proliferation with respect to the metabolic assay in comparison to DNA binding fluorophores.

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.