New Approaches To Intracellular Analysis

The Tsien group has developed several exciting new approaches to in situ analysis. Miyawaki et al. (1997) introduced the use of Green Fluorescent Protein variants fused to calmodulin, such that the dramatic conformational change induced in the dumbbell-shaped protein by binding calcium was transduced as a change in energy transfer efficiency (Figure 9.1). This approach combines the advantages of energy transfer assays with an expressible indicator To measure the analyte, all one need do is...

Fluorescence Detection For Dna Sequencing

Following the electrophoretic separation of DNA sequencing fragments, they need to be detected and identified. Since the first demonstrations of the use of fluorescence tags to label DNA oligonucleotides for sequencing (Ansorge et al., 1986, 1987 Prober et al., 1987 Smith et al., 1986), fluorescence detection has been accepted as the predominant detection protocol for DNA sequencing. It has allowed DNA sequencing to be performed in an automated fashion with base-calling performed during the gel...

Dna Surface Recognition

Multifunctional fabricated nanoparticles can be utilized as a receptor for recognition of biomacromolecular surfaces such as proteins, peptides, and DNA. Among all of the macro-molecular surfaces, DNA is considered as a relatively simple surface for receptor targeting. In spite of its simplicity, the enormous specificity of the Watson-Crick hydrogen bonding allows for convenient programming of artificial DNA receptors. Another very attractive feature of DNA is the great mechanical rigidity of...

Overview Of Spri Methodology

Surface plasmon resonance (SPR) is a surface-sensitive optical technique that can be applied to the real-time monitoring of biomolecular adsorption and or desorption events at biopolymer layers formed on a thin gold film or other noble metal surfaces. Surface plasmons are electromagnetic waves that propagate along a metal dielectric interface. The optical field intensity of the surface plasmon waves decays exponentially from the surface of the metal into the dielectric layer. For a gold film,...

Direct Detection Of Dna By Hybridization Adsorption

The direct analysis of genomic DNA and RNA in an array format without labeling or PCR amplification would be extremely advantageous for a variety of biological applications. The detection and identification of ssDNA oligonucleotides by sequence specific adsorption onto an ssDNA microarray element to form a double-stranded duplex is called hybridization adsorption. If the target oligonucleotide is labeled with a fluorophore, fluorescence imaging can be used to detect DNA with the microarray. If...

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Live-cell detection of RNA molecules using the MS2 system. RNA molecules are detected using a fusion of green fluorescent protein (GFP) with the MS2 bacteriophage coat protein,which has an extremely high affinity for a short RNA-recognition motif that is derived from the phage genome. Single mRNA molecules that contain 24 MS2 RNA repeats can be detected. Adapted from Shav-Tal et al. (2004b). See insert for color representation of this figure. Figure 2.3. Live-cell detection of RNA...

Living Cells References

Agrawal, A, Zhang, C, Byassee, T, Tripp, RA, and Nie, S (2006). Counting single native biomolecules and intact viruses with color-coded nanoparticles. Anal Chem 78(4) 1061-1070. Ambrose, WP, Goodwin, PM, Jett, JH, Orden, AV, Werner, JH, and Keller, RA (1999). Single molecule fluorescence spectroscopy at ambient temperature. Chem Rev 99 2929-2956 and references therein. Axelrod, D, Burghardt, TP, and Thompson, NL (1984). Total internal reflection fluorescence. Ann Rev Biophys Bioeng 13 247-268....

Nanoscale Manipulations Of Subcellular Compartments And Chemical Reactions Of Their Contents

Effects of Diffusion and the Need for Small Reaction Volumes To manipulate chemically (e.g., dye tagging the metabolites) the contents of a nanometer-sized subcellular compartment, the main challenge is to overcome dilution by spatially confining the chemical reaction to an ultrasmall volume. Figure 6.6 is a simple estimation that shows the importance of spatially localizing the reactants and the rapid nature of diffusion at the nanometer and micrometer length scale. Figure 6.5....

References

Electrogenerated chemiluminescence. 40. A chemiluminescent polymer based on the system. J Am Chem Soc 104 2641-2642. Alivisatos, A 1996 . Perspectives on the physical chemistry of semiconductor nanocrystals. J Phys Chem 100 31 13226-13239 and references therein. Arora, A, Eijkel, JCT, Morf, WE, and Manz, A 2001 . A wireless electrochemiluminescence detector applied to direct and indirect detection for electrophoresis on a microfabricated glass device. Anal Chem...

Referencesvtvlivingcells

Abad, JM, Mertens, SFL, Pita, M, Fernandez, VM, Schiffrin, DJ 2005 . Stereochemical recognition of doubly functional aminotransferase in 2-deoxystreptamine biosynthesis. J Am Chem Soc 127 5689-5694. Blow, MD 1976 . Structure and mechanism of chymotrupsin. Acc Chem Res 9 145-152. Boal, AK, and Rotello, VM 2000a . Fabrication and self-optimization of multivalent receptors on nanoparticle scaffolds. J Am Chem Soc 122 734-735. Boal, AK, and Rotello, VM 2000b . Intra- and intermonolayer hydrogen...