Amplification and Restriction for Methylation Determination

DNA methylation is a key epigenetic mechanism for gene regulation during development and carcinogenesis, and the methylated DNA of genetic loci associated with the latter are increasingly being exploited as useful biomarkers for cancer. For typical clinical samples such as blood and urine, this requires the sensitive detection of very small amounts of methylated DNA from a tumor in a high background of unmethylated DNA from normal tissue. While there are several PCR-based assays for this purpose, such as methylation-specific PCR (MSP) of bisulfite-treated sample DNA, these may not work well for certain markers due to difficulties in primer design. As a complement to these techniques, C. Kneip, D. Dietrich, and their colleagues at Epigenomics AG (Berlin, Germany) as well as others at the Charité-Universitätsmedizin (Berlin, Germany) have devised a PCR assay that specifically and sensitively amplifies methylated DNA through the digestion of bisulfite-converted, unmethylated DNA using the heat-stable restriction enzyme Tsp509I during the PCR reaction itself. The assay depends on the presence of the pentanucleotides AATCG or AACCG in the target sequence, which in their unmethylated form are both bisulfite-converted to AATTG, which contains the Tsp509I cleavage site AATT, whereas the methylated form of both pentanucleotides are bisulfite converted to AATCG. Therefore, during each PCR cycle, Tsp509I will digest any unmethylated DNA, while the methylated DNA is not digested and will be amplified. Using the lung cancer DNA methylation biomarker BARHL2 (Bar-like homeobox), they first validated their assay on samples with defined ratios of methylated and unmethylated DNA, and then demonstrated that its clinical performance on 75 bronchial lavage samples was comparable to that of MSP.

(See “A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling”)

To Dye With a Halo

Proper cellular function depends on localized pH and its modulations, yet details of pH differences within cells remain elusive due to a paucity of intra-cellular pH sensors needed for research. At a minimum, sensors must have pKa values within the pH range of the cellular compartment of interest and show distinct excitation or emission wavelengths corresponding to their protonated and unprotonated states. Currently, the majority of organic fluorescent pH-sensitive dyes act as global indicators rather than reporting the pH of localized subcellular regions. Protein-based pH indicators have been engineered to detect the pH of subcellular compartments, but with the success of achieving localized values came several disadvantages, including the requirement for dual-excitation, a limited dynamic range, the necessity for excitation in a part of the spectrum known to promote noise, or the need for extensive experimental optimization. H. Benink, G. Los, and their colleagues at Promega Biosciences (Madison, WI) combined a protein-based reporter system (HaloTag) with a well-established organic dye (SNARF-1) to measure intracellular pH. Increasing the linker length between the two components allowed sufficient separation of the protein and the dye, resulting in a pH sensor that mimicked the behavior of the original dye under optimal conditions. The resulting product demonstrated subcellular targeting of the sensor and accurate determination of the pH of the target regions when used in ratiometric confocal microscopy. The chemistry needed to attach the HaloTag to a sensor is simple, allowing easy exploitation of the technology for other needs.

(See “Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells”)

A Green Light to Titrate Baculovirus

The expression of eukaryotic proteins using the baculovirus system has been a great benefit to molecular biologists, permitting the expression of larger proteins at higher expression levels with the appropriate post-translational modifications when compared to using prokaryotic expression systems. Titration of recombinant baculovirus stocks is an essential procedure when carrying out infections for protein production, in order to ensure the optimal level of expression. While various methods have been developed to quantitate baculovirus particles, these suffer various drawbacks: molecular assays such as qPCR or anti-gp64 antibody assay measure total and not infectious baculo-virus, while infection-based techniques such as plaque assay and end-point dilution for cytopathic effect can be tedious and lengthy. In order to facilitate the scoring of the end-point dilution assay, which is less labor-intensive than the plaque assay, marker genes such as β-gal and GFP have been incorporated into the baculovirus vector, but this requires that the protein of interest be cloned in these specific vectors in order to be titrated and results in co-expression of the marker protein, which may not be desirable. To overcome these disadvantages, R. Hopkins and D. Esposito at the National Cancer Institute (Frederick, MD) have devised a novel cell line containing an eGFP gene under the control of the baculo-virus polyhedron late promoter. Infection of this cell line with a baculovirus and subsequent expression of early baculovirus proteins induces GFP expression through activation of the polyhedrin promoter. Small foci of infected GFP-expressing cells are easily visible under a fluorescent microscope, and using a 96-well end-point dilution assay the titer can be easily determined in three days as opposed to seven days for the cytopathic effect, which is also more difficult to score.

(See “A rapid method for titrating baculo-virus stocks using the Sf-9 Easy Titer cell line”)