Technical challenges in applying capillary electrophoresis-single strand conformation polymorphism for routine genetic analysis

Recent and future advances in population genetics will have a significant impact on health care practices and the economics of health care provision only if a spectrum of patient-tailored, effective methods of DNA screening for sequence alterations has been developed. Genetic screening by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP), which is based upon the differences in electrophoretic mobilities of wild-type and mutant DNA species, offers an important complement to other presently available techniques such as Sanger sequencing and DNA hybridization arrays due to its simplicity, versatility, and low cost of analysis. A two-part review of CE-SSCP that discusses its advantages and limitations is presented. Emphasis is placed on technological aspects of CE-SSCP (including such rarely addressed issues as sample preparation protocols and the nature of the polymeric DNA separation matrix) as well as on the potential of CE-SSCP for routine genetic analysis. An attempt is made to organize and present the information in sufficient detail to allow the use of SSCP for routine genetic screening even by those inexperienced in CE. Some discussion of CE-based heteroduplex analysis (HA) is also presented.     

Formation of 7,9-Disubstituted Xanthinium Compounds Under Mild Conditions by Alkylation of Silylated Xanthines

Silylated, 3-unsubstituted xanthine derivatives afford 7,9-diallyl and 7,9-dibenzylxanthinium compounds upon the reaction with excess 2-propenyl (allyl) or benzyl bromide at room temperature.     

Donor-Acceptor Substituted Cyclohexasilanes: Materials with Potential Nonlinear Optical Properties

Summary.  The dipolar cyclohexasilanes 1-ferrocenyl-4-((2,2-dicyanoethenyl)-phenyl)-decamethylcyclohexasilane (1) and 1-ferrocenyl-3-((2,2-dicyanoethenyl)-phenyl)-decamethylcyclohexasilane (2) were synthesized by conventional methods and fully characterized. Electronic absorption spectroscopic studies suggest increased σ-π interactions in the cyclohexasilane derivatives when compared to their open-chained analogues. Small but noticeable electron transmission effects via the cyclohexasilanyl group can be derived from cyclic voltammetric data. Significantly increased values of the quadratic hyperpolarizabilities β in solution for 1 and 2 are shown by preliminary results of measurements using the EFISH technique. Received May 30, 2000. Accepted December 20, 2000     

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.     

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.     

Modular Synthesis and Electronic and Hole-Transport Properties of Monodisperse Oligophenothiazines

Summary: Monodisperse oligophenothiazines are rapidly synthesized in good yields by use of Suzuki arylation. These well defined oligomers reveal both highly fluorescence and low oxidation potentials. As a consequence of intense electronic coupling as supported by cyclic voltammetry a selected trimer shows hole-transport properties in a hole-conductor-only set-up of a diode with relatively low on-set voltage.     

Ferroelectric liquid crystals induced by dopants with axially chiral 2,2'-spirobiindan-1,1'-dione cores: diether vs. diester side-chains

A series of axially chiral 5,5'- and 6,6'-dialkanoyloxy-2,2'-spirobiindan-1,1'-dione dopants, (R)-2 and (R)-4a-4c were synthesized in optically pure form and their ferroelectric polarization powers, δ_p, measured in four liquid crystal hosts with isotropic (I)-nematic (N)-smectic A (SmA)-smectic C (SmC) phase sequences. The results show that the sign of polarization P _S induced by (R)-2 and (R)-4a-4c follows the same trend as that previously reported for the 5,5'- and 6,6'-diheptyloxy-2,2'-spirobiindan-1,1'-dione dopants, (R)-1 and (R)-3. The polarization induced by (R)-2 in the host DFT is below detection limits, and the sign of P _S was found to invert as a function of temperature at mole fractions as low as 0.01. On the other hand, the polarization power of the 6,6'-diheptanoyloxy dopant (R)-4b in the host NCB76 is -1449 nC cm^-2, the fourth highest value reported so far, and more than three times the δ_p value of the 5,5'-diheptanoyloxy analogue (R)-2 in that host (+474 nC cm^-2). Results of ²H NMR experiments suggest that (R)-4b exerts stronger local perturbations in NCB76 than (R)-2, and that these perturbations may be chiral in nature.     

Protein-based radicals in the catalase-peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site

Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.