‘Dual’-ing Emulsion PCR Methods

Emulsion PCR is utilized in several next-generation sequencing methods to clonally amplify a DNA fragment onto a single bead prior to pyrosequencing or sequencing- by-ligation (SBL) of one end of the amplicon. Because these methods tend to produce short sequence reads, paired-end sequencing was devised to optimize their use in applications such as the analysis of transcriptomes and genomic rearrangements. In this strategy, short tags corresponding to both ends of a DNA fragment are extracted and ligated together to form a paired-end ditag, which is then sequenced. Comparison of the ditag sequence to a reference genomic sequence delimits the ends of the original DNA fragment from which the ditag is derived. Generating paired-end ditags for sequencing, however, can be time-consuming and labor- intensive. J. Edwards and colleagues at the University of New Mexico (Albuquerque, NM) have developed a new approach for paired-end sequencing using emulsion PCR called dual primer emulsion PCR (DPePCR), which dispenses with the need for ditags. In their technique, both the forward and reverse primers are attached to the beads, in contrast to the single primer used in the standard protocol. Emulsion PCR in the presence of one bead, both primers in solution, and PCR reagents results in the efficient amplification of a single DNA molecule after 120 cycles into multiple bridged amplicons on the bead. Digestion at type IIS restriction enzymes sites located in the adapter sequences at the ends of the bridged DNA amplicons leaves the short sequences from both ends attached to the bead. The free ends of these tags are then ligated to capping adapters containing anchor primer binding sites. Using the appropriate anchor primers, both ends of the starting DNA fragment can be sequenced by SBL in either the 5′ to 3′ or 3′ to 5′ direction. This simple modification will allow faster and less expensive paired end sequencing using emulsion PCR–based next-generation sequencing.

Schematic diagram of the emulsion PCR step in one droplet for the DPePCR method. (Click to enlarge)

See “Dual primer emulsion PCR for next-generation DNA sequencing”.

Simple Sample Tracking

Microarray technology has become increasingly popular for whole-genome expression profiling. While in many cases, experimental designs have been refined and quality control measures implemented at each step of the procedure, there is still the potential for error, especially when dealing with large numbers of samples and arrays. One of the most important considerations is maintaining sample identity throughout many steps where samples are treated and transferred between tubes. Confusion of samples can lead to erroneous results or complete failure of a microarray study. To track samples, some labs have implemented laboratory information management software systems, but these systems are still open to human error and may not be available in smaller laboratories or to researchers running few microarray experiments. In this issue of BioTechniques, M. Walter and colleagues at the University of Tübingen (Baden-Württemberg, Germany) present an RNA sample tracking system for use in microarray analysis. The authors take advantage of a set of probes complementary to bacterial sequences that are found on Affymetrix chips to generate eight different spike-in controls. When added into RNA samples in different combinations, the control sequences hybridize to the probes present on the arrays, resulting in 256 possible sample-specific hybridization patterns that can be translated into eight-digit binary numbers to barcode the RNA present on the array. To determine if the spiked-in controls influenced expression patterns observed on the arrays, the authors hybridized side-by-side arrays with the same RNA, either with the added controls or without. When plotted against each other, the normalized signals from the arrays correlated within the range of technical replicates, indicating that the spike-in controls did not influence expression levels for the remaining probe sets on the array. The authors went on to demonstrate that control samples had no negative effect on RNA integrity or impact on the overall performance of the microarray experiment. This barcoding approach is inexpensive and adaptable since Illumina arrays also harbor control probes and custom arrays can be easily modified to carry such probes.

See “Utilization of AFFX spike-in control probes to monitor sample identity throughout Affymetrix GeneChip array processing”.

Nephelometry for Filamentous Fungi Phenotyping

Whole-genome sequences are available for more than 100 fungal species, yet the functions of many proteins encoded by those genomes remain undiscovered. To facilitate protein function analysis, deletion strains for each Saccharomyces cerevisiae and Neurospora crassa gene have been collected and made available to interested researchers. These strain collections, along with microscale liquid cultivation and automated growth recording, have enabled high-throughput phenotypic profiling of yeast. But these systems are not optimal for studying filamentous fungi. Spectrophotometric assays measuring the transmission of light aid in automated growth recording for single-cell organisms and are sometimes employed to monitor the growth of filamentous fungi as well, but the accuracy of the readings can be impaired by hyphae clumping and non-homogeneous growth of fungi in liquid media. With filamentous fungi, quantitative phenotypic data must be obtained using time-consuming microscopic measurements or assays of expansion rates of colonies growing on solid media, methods that are not amenable to high-throughput screens. These methods also do not deliver data regarding the specific effects of a mutation or environmental change on growth, such as changes in the growth rate of the fungi. A. Joubert and colleagues at Université Faculté des Sciences (Angers, France) developed a new method for automated monitoring of filamentous fungal growth using nephelometry, which measures the scattering of light to detect the opacity of media. Their method depends on a commercially available laser-based microplate nephelometer, as well as microscale liquid cultivation. The authors found a linear relationship between the number of cells and nephelometric values at all cell densities tested, indicating that raw values can be used directly for growth curve analysis without the need for data conversion. Dry weight mycelial mass measurements and microscopic examination of the hyphae and branching numbers collected at specific growth intervals were highly correlated with changes in nephelometric measurements, signifying that laser nephelometry can accurately indicate fungal biomass and is a reliable means of monitoring fungal growth. The system also facilitated large-scale phenotypic profiling as demonstrated by testing known antifungal compounds on wild-type and mutant Alternaria brassicicola.

See “Laser nephelometry applied in an automated microplate system to study filamentous fungus growth”.

High-er EdU-cation

High-throughput screens for potential therapeutic agents targeting diseases that affect cell growth or division depend on assays that measure cell proliferation. Many of these are endpoint assays that measure cell number by determining metabolic activity, ATP content, or cellular biomass. However, while cell number partially indicates cell proliferation, it cannot distinguish effects on cell death from cell proliferation, nor does it indicate the cell cycle phase of samples. Proliferation can be more accurately determined by measuring DNA replication through incorporation of labeled nucleotides, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA. BrdU incorporation can be detected by flow cytometry or microscopy, enabling the measurement of DNA synthesis in individual cells of a heterogeneous population. But this approach requires a monoclonal anti-BrdU antibody and antigen access induced by heat, acid, or nuclease treatment, which can be harsh on the samples and expensive for high-throughput screening applications. As an alternative, T. Gonda and colleagues at University of Queensland (Brisbane, Australia) have adapted the cell proliferation assay that is based on the incorporation of the novel nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). The alkyne-substituted nucleoside incorporated in DNA can be coupled with azide-substituted fluorescent dyes through a copper-catalyzed cycloaddition (CuAAC or “click ”) reaction that is rapid, highly specific, and involves minimal sample treatment. This less expensive protocol does not require commercial kits or custom reagents and is fully automated to work with 96-well plates in a high-throughput liquid handing system. Treated samples are analyzed in situ by high-content fluorescent microscopic imaging. The authors validated their method by assaying the proliferation of MCF10A mammary epithelial cells grown in various serum concentrations with or without epidermal growth factor (EGF), as well as treating the cells with various inhibitors of protein kinases in the EGF signaling pathway. When coupled with the DNA stain 4,6′-diamidino-2-phenylindole (DAPI), this adaptation of the EdU incorporation assay for DNA synthesis was demonstrated to be well-suited for analysis of cell proliferation in a high-throughput format.

Fluorescence micrograph of DAPI-stained nuclei with the Cy5 adduct of the incorporated nucleoside analog EdU. (Click to enlarge)

See “Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening”.