In Tune

Gold and silver nanoparticles (NPs) are exceptionally photo-stable, can be targeted via biofunctionalization, and offer high spatial resolution in cell-imaging applications. But, for all their advantages, NPs can at times be difficult to distinguish from small vesicles and similar intracellular features and cannot probe multiple targets in the same sample. In Analytical Chemistry, Sun et al. show how tuning the wavelength of differential interference contrast (DIC) microscopy when imaging NPs may address these concerns. Based on the properties of DIC microscopy, NPs are detectable within a relatively narrow range of illumination wavelengths, centered around 540 and 420 nm for gold and silver NPs, respectively. A quick switch between wavelengths in which the NPs are and are not visible lets the user determine whether or not an ambiguous signal truly derives from a NP probe. But for those willing to modify their DIC microscopes, Sun et al. describe simultaneous imaging at 540 nm (picking up gold NPs and cellular components) and 720 nm (cellular material only). Because DIC microscopy enables finely detailed imaging without overly stressing cells, the technique is also applicable to single-particle tracking, as Sun et al. show by monitoring the endocytosis of a Tat peptide–coated gold NP. Although the authors do not demonstrate dual imaging of gold and silver NPs in cells, they report a successful proof-of-principle experiment where each type of NP was imaged after it had been conjugated to a particular DNA oligo-nucleotide and hybridized to cognate sequences on a glass slide. The ability to reliably distinguish gold and silver NPs suggests many potential uses for differently functionalized probes in multi-target labeling and tracking experiments.

Reprinted with permission © 2009 ACS. (Click to enlarge)

Sun et al. Wavelength-dependent differential interference contrast microscopy: selectively imaging nanoparticle probes in live cells. Anal. Chem. [Epub ahead of print, September 29, 2009, doi: 10.1021/ac901623b].

How Low Can You Go?

Even as genomics facilities rush to finish a representative collection of complete human genome sequences, targeted resequencing efforts remain an important means of uncovering rare disease-associated variants. For those labs pursuing targeted resequencing approaches, squeezing the maximum data out of each instrument run remains a central concern. A recent paper in Human Mutation from Out et al. examines the question of the minimum allele frequency that can be reliably detected from pooled samples analyzed using Illumina's parallel sequencing platform. A first consideration is whether samples should be pooled before or after long-range PCR. Although the latter is often standard practice, Out et al. found that this compromised sensitivity when they sequenced mutY homolog (Escherichia coli) (MUTYH) as a candidate gene in samples from breast cancer patients. The authors contend that pre-PCR pooling is only advisable for genomic DNA expected to be of homogenous quality; for many studies involving clinical samples collected at different times and by varied means, differences in DNA quality will likely lead to skewed amplification. Using a post-PCR pool of 88 samples that had previously been subjected to traditional Sanger sequencing methodology, Out et al. resequenced to a calculated sequencing depth of ~330 reads per base pair per sample. This level of coverage was sufficient to pick up all doubleton (homozygous) variants, although only about half of the singleton variants were detected. The authors conclude that Illumina users can expect to detect polymorphisms at a 0.5% minor allele frequency, but also provide analyses of sequence depth–statistical power correlations that should help researchers planning targeted resequencing to estimate a reasonable pool size for subsequent optimization.

Out et al. Deep sequencing to reveal new variants in pooled DNA samples. Hum. Mutat. [Epub ahead of print, September 4, 2009, doi: 10.1002/humu.21122].

Coloring Outside the Lines

Real-time PCR amplification is generally monitored by one of two basic paradigms: molecular beacons that un-quench as the reaction progresses, or an intercalating dye that signals increasing amplicon concentration. However, this distinction is being blurred by a new approach pioneered by Takei et al. in Angewandte Chemie. Like molecular beacon approaches, the new method relies upon oligonucleotide probes, but does not require covalent attachment of a fluorophore and quencher. Instead, Takei and colleagues took advantage of a molecule (2,7-diamino-1,8-naphthyridine, or DANP) with an affinity for DNA hairpins containing a bulged C nucleotide. Within the bulge, DANP exists in its protonated form and fluoresces maximally at 430 nm; once released, its fluorescence maximum shifts to 400 nm. By tagging a PCR primer with a suitable bulged hairpin and monitoring the fluorescence signal specific to the bulge-bound DNAP during the amplification reaction, the authors were able to show the progress of the reaction as the primer hairpins melt in order to anneal with the newly synthesized complementary strand. Takei et al. report that a 28-nucleotide bulged hairpin tag works with a variety of primer sequences to signal DANP release over the course of PCR. In addition, the DANP-binding tag can be used in allele-specific amplifications to selectively detect sequence variants. This hybrid technique may offer a convenient way to combine the specificity of molecular beacon detection with the low cost and convenience of unincorporated fluorescent dyes.