A major focus of our lab is the early detection of prostate cancer. Many recent studies show a change in the methylation status of specific genes in cancer cells. The level of methylation at CpG sites is measured by QPCR (see below). Using this method, detection of cancer has the possibility of being done faster and with less trauma to the patient. It also has the potential to be more accurate (more true positives and less false negatives) than with the PSA test, leading to more correct diagnosis and less unnecessary biopsies.
Our lab uses the Corbett Research Rotor-Geneä RG3000 to perform Quantitative PCR (QPCR). In our research with DNA methylation, we use Methylation Specific PCR (MSP) to determine the methylation status of CpG islands of genes thought to indicators of cancer. For this procedure, genomic DNA from a patient is treated with bisulfite to convert unmethylated cytosine residues into uracil. Methylated cytosine (5mCytosine) remains unreacted. Primers that distinguish between cytosine and uracil at CpG sites are used to amplify the target in separate reactions. Taqman probes specific to the bisulfite converted sequence between the primers allow for high specificity of detection by fluorescence.
DNA Sizing (Agilent)
The Agilent Bioanalyzer 2100 is a useful tool in the lab for quickly analyzing various DNA samples quantitatively. The DNA LabChipâ electrophoretically separates DNA based on size through microchannels in about 30 minutes with only 1ml of input. PCRs can be quickly checked for amplification of appropriately sized products or for the presence of primer-dimers. We have used the Bioanalyzer to develop an EMSA that can check for Protein-DNA complexes based on mobility shift.
A Waters HPLC System with a 996 Waters Photodiode Array Detector is used in the lab for oligomer (TAQMAN probes) purification. The purifications are performed on a PRP-1 column, using reversed-phase chromatography, in TeBAA (50 mM tetrabutylammonium acetate buffer, adjusted to pH 7.0 with acetic acid, in a gradient of acetonitrile), and TEAA buffer (50 mM triethylammonium acetate buffer, adjusted to pH 7.0 with acetic acid in a gradient of acetonitrile)
A Cyclone Plus DNA Synthesizer is used in the lab to synthesize oligos such as PCR primers, TAQMAN probes and substrates for enzyme kinetics experiments.
DNA and RNA quantification is done using a Nanodrop ND-1000 Spectrophotometer. “The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of extremely small samples with remarkable reproducibility. The patented sample retention system eliminates the need for cuvettes and capillaries which decreases the measurement cycle time. In addition, the high absorbance capability eliminates the need for most dilutions.” Quantifications are also performed using a Gilford Response spectrophotometer and a GeneQuant Pro RNA/DNA Calculator.