Richard Williams, Peisajovich, Sergio G, Miller, Oliver J, Magdassi, Shlomo , Tawfik, Dan S, and Griffiths, Andrew D. 2006.
“Amplification Of Complex Gene Libraries By Emulsion Pcr.”. Nat. Methodsnature Methods, 3, Pp. 545 - 550.
Abstract The efficient amplification of genomic libraries, cDNA libraries and other complex mixts. of genes by PCR is impeded by two phenomena: firstly, short fragments tend to be amplified in preference to larger ones; and, secondly, artifactual fragments are generated by recombination between homologous regions of DNA. Recombination in this case occurs when a primer is partially extended on one template during one cycle of PCR and further extended on another template during a later cycle. Thus, chimeric mols. are generated, the short ones of which are then preferentially amplified. A variety of PCR protocols have been proposed to minimize these problems, most of which rely on high template concns. and low nos. of PCR cycles. Clearly, however, such an approach is not viable if little template DNA is available. Here we describe a protocol for amplifying complex DNA mixts., based on the compartmentalization of genes in a water-in-oil (w/o) emulsion. Template fragments are segregated in the minute aq. droplets of the emulsion and amplified by PCR in isolation. This approach alleviates the problems described above while enabling the use of small amts. of template DNA and high nos. of PCR cycles. Box 1 described an alternative method for generating very stable emulsions for emulsion PCR using the surfactant ABIL EM 90. [on SciFinder(R)]
Oliver J Miller, Bernath, Kalia , Agresti, Jeremy J, Amitai, Gil , Kelly, Bernard T, Mastrobattista, Enrico , Taly, Valerie , Magdassi, Shlomo , Tawfik, Dan S, and Griffiths, Andrew D. 2006.
“Directed Evolution By In Vitro Compartmentalization.”. Nat. Methodsnature Methods, 3, Pp. 561 - 570.
Abstract The goal of in vitro compartmentalization (IVC) is to divide a large reaction between many microscopic compartments. This technique was first developed to generate "artificial cells" for the directed evolution of proteins. Typically, an aq. soln. of genes and an in vitro transcription-translation system is stirred (or homogenized) into an oil-surfactant mixt. to create a water-in-oil (w/o) emulsion with -1010 aq. droplets per mol of emulsion. The majority of droplets contain no more that a single gene along with all of the mol. machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype in vitro, making it possible to select very large libraries of genes (108-1011 genes). We describe the advantages and applications of IVC. A protocol for performing a directed evolution expt. by IVC makes use of one or more w/o emulsions. This procedure involves the generation of a gene library, the performance of a selection, and the subsequent recovery of the selected genes by PCR. We also describe two procedures for converting w/o emulsions to water-in-oil-in-water (w/o/w) emulsions for high-throughput screening using a fluorescence-activated cell sorter (FACS). Finally, we describe two methods for delivering substrates, regulators and other compds. to the preformed aq. droplets of w/o emulsion. [on SciFinder(R)]
Pure magnetite nanoparticles (Fe3O4) have been synthesized in water by copptn. using two different approaches (from ferrous sulfate and a mixt. of ferrous and ferric chlorides). All materials aggregated in aq. suspension, but their subsequent dispersion on treatment with a variety of agents was obsd. to be different. Magnetite produced using ferrous sulfate could not be disaggregated, whereas magnetite produced from a mixt. of ferrous and ferric chlorides could be disaggregated to a quasi-monodispersed form. The dispersing agents were tetra-Me ammonium hydroxide, Disperbyk 190 and polyacrylic acid. The finding has potentially important implications for the surface activation of superparamagnetic magnetite nanoparticles and their ability to be used in bio/life science applications. [on SciFinder(R)]
Nanoparticles of metals having high elec. cond. offer new scope for direct digital printing of conductive patterns. Stable aq. silver nanoparticle dispersions were synthesized by chem. redn. These were suitable for inkjet printing, with the dried inks sintering at temps. as low as 160°. However, the resistance remained much higher than that of metallic silver. [on SciFinder(R)]