Search PubMed⌕ Search

Biomedical subjects

R A Mathies

Publications and source records attributed to R A Mathies.

At least 19 recordsLinked to original sources
PubMed1999

Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration.

Chiral separations of fluorescein isothiocyanate-labeled amino acids have been performed on a microfabricated capillary electrophoresis chip to explore the feasibility of using such devices to analyze for extinct or extant life signs in extraterrestrial environments. The test system consists of a folded electrophoresis channel (19.0 cm long x 150 microns wide x 20 microns deep) that was photolithographically fabricated in a 10-cm-diameter glass wafer sandwich, coupled to a laser-excited confocal fluorescence detection apparatus providing subattomole sensitivity. Using a sodium dodecyl sulfate/gamma-cyclodextrin pH 10.0 carbonate electrophoresis buffer and a separation voltage of 550 V/cm at 10 degrees C, baseline resolution was observed for Val, Ala, Glu, and Asp enantiomers and Gly in only 4 min. Enantiomeric ratios were determined for amino acids extracted from the Murchison meteorite, and these values closely matched values determined by HPLC. These results demonstrate the feasibility of using microfabricated lab-on-a-chip systems to analyze extraterrestrial samples for amino acids.

Amino Acids↗
PubMed1999

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Base Sequence↗
PubMed1999

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis.

We have constructed a rotary confocal fluorescence scanner and capillary array electrophoresis system that is designed to analyze over 1000 DNA sequencing or fragment sizing separations in parallel. Capillaries are arranged around the surface of a cylinder and a rotating objective in the middle of the cylinder excites and collects fluorescence from labeled DNA fragments as they pass the capillary detection window. The capillaries are pressure-filled with a replaceable matrix and the samples are electrokinetically injected in parallel from a stainless steel microtiter plate at the cathode end. We demonstrate that the instrument is capable of producing four-color data from all capillaries at a scan rate of 4 Hz (corresponding to a linear scan velocity of 121 cm/s). M13 sequencing data were obtained using a 128 capillary array mounted in half of the first quadrant of the scanner. In this initial run, read lengths greater than 500 bases were obtained in over 60% of the capillaries.

Bacteriophage M13↗
PubMed1999

How color visual pigments are tuned.

The absorption maximum of the retinal chromophore in color visual pigments is tuned by interactions with the protein (opsin) to which it is bound. Recent advances in the expression of rhodopsin-like transmembrane receptors and in spectroscopic techniques have allowed us to measure resonance Raman vibrational spectra of the retinal chromophore in recombinant visual pigments to examine the molecular basis of this spectral tuning. The dominant physical mechanism responsible for the opsin shift in color vision is the interaction of dipolar amino acid residues with the ground- and excited-state charge distributions of the chromophore.

Color Perception↗
PubMed1998

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants.

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.

Amino Acid Sequence↗
PubMed1998

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Capillary Action↗
PubMed1998

Capillary electrophoresis chips with integrated electrochemical detection.

Capillary electrophoresis systems with integrated electrochemical detection have been microfabricated on glass substrates. Photolithographic placement of the working electrode just outside the exit of the electrophoresis channel provides high-sensitivity electrochemical detection with minimal interference from the separation electric field. Microchip electrophoretic separations of neurotransmitters in under 100 s exemplify the good resolution and attomole detection sensitivity of these devices. Using indirect electrochemical detection, high-sensitivity DNA restriction fragment and PCR product sizing has also been performed. These microdevices match the detector's size to that of microfabricated separation and reaction devices, bringing to reality the lab-on-a-chip concept.

DNA↗
PubMed1998

Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations.

Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence emission. Primers with the following donor/acceptor pairs were synthesized: 6-carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine/ 6-carboxy-X-rhodamine (CYA-ROX), and the 4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3-propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) BODIPY (BODIPY503/512-BODIPY581/591). Variables examined included the length of the 5'-amino linker arm, the number of base pairs between the donor and acceptor, and the excitation wavelength (488 or 514 nm). Of the primers examined, CYA-ROX primers offer the best combination of acceptor fluorescence emission intensity and spectral purity.

Amino Acids↗
PubMed1998

A three-wavelength labeling approach for DNA sequencing using energy transfer primers and capillary electrophoresis.

Capillary electrophoresis DNA sequencing has been accomplished by using four different energy transfer primers and three fluorescence detection channels. Methods have also been developed to deconvolve the three-color data into the four base concentrations. The nonnegative least squares and model selection method resulted in the best accuracy. The three-color data were compared to sequencing data obtained using four detection channels and four energy transfer primers. The average accuracy rates obtained over three 500 base M13mp18 runs using three-color coding were 96% including 18 uncallable compressions and 99.6% if these compressions are excluded. The average accuracy rate obtained using four-color coding was 96.3% including 18 uncallable compressions and 99.9% if these compressions are excluded. Although it is unlikely that three-color schemes will replace four-color sequencing, these methods have exposed basic concepts that will be useful in the development of higher-order multiplex coding methods for DNA analysis.

DNA Primers↗
PubMed1997

Ultraviolet resonance Raman examination of the light-induced protein structural changes in rhodopsin activation.

Ultraviolet resonance Raman (UVRR) spectra of rhodopsin and its metarhodopsin I and metarhodopsin II photointermediates have been obtained to examine the molecular mechanism of G-protein-coupled receptor activation. Spectra were acquired using a single-pass capillary flow technique in combination with a Littrow prism UV prefilter detection system. The UVRR difference spectra between rhodopsin and mearhodopsin I exhibit small differences assignalbe to tyrosine residues and no differences due to tryptophan. The UVRR difference spectra between rhodopsin and metarhodopsin II exhibit significant differences for vibrations of both tryptophan and tyrosine residues. Most importantly, there is and intensity decrease of the totally symmetric tryptophan modes at 759, 1008, and 1545 cm-1, an intensity decrease of the tryptophan W7 band at 1357 cm-1, and a frequency shift of the tryptophan W17 ban from 885 to 892 cm-1. These difference features are assigned to one or more tryptophan residues that reside in a hydrophobic, weakly hydrogen-bonding environment in rhodopsin and that are transferred to a less hydrophobic, non-hydrogen-bonding environment during rhodopsin activation. The available evidence suggests that Trp265 makes a dominant contribution to the tryptophan features in this difference spectrum. These results are interpreted with a model for rhodopsin activation in which retinal isomerization alters the interaction of Trp265 with the ionone ring of the retinal chromophore.

Animals↗
PubMed1997

Optimization of spectroscopic and electrophoretic properties of energy transfer primers.

We have synthesized and characterized the spectroscopic properties of 56 energy transfer (ET) fluorescent dye-labeled primers differing in (i) the spacing between the donor and acceptor, (ii) the nature of the spacer (either oligonucleotide or polydideoxyribose phosphate), (iii) the primer sequence (M13 (-40), M13 (-21), M13 reverse, SP6, T3, and T7 priming sequences), and (iv) the dyes chosen as the donor (6-carboxyfluorescein, F; or 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine, C) and acceptor (F; 5 & 6-carboxyrhodamine-110, R110; 6-carboxyrhodamine-6G, G; N,N,N',N'-tetramethyl-6-carboxyrhodamine, T; and 6-carboxy-X-rhodamine, R) chromophores. This study led to the development of two significantly improved ET primer sets for multiple-color analyses. These primers are named using the convention D-N-A, where D is the donor, A is the acceptor, and N is the number of nucleotides between the donor and the acceptor. The primer set C4R110, C4G, C4T, and C4R provides acceptor emissions of high spectral purity with donor:acceptor emission ratios of < 0.002 for C4G, < 0.004 for C4T, and < 0.005 for C4R and excellent matching in the electrophoretic mobilities of single-base extension DNA fragments. The C4R110, C4G, C4T, and C4R set is valuable for diagnostic applications where minimization of crosstalk between different labels is of particular importance. The set C10R110, C10G, C10T, and C10R, which uses only rhodamine dyes as acceptors, shows significantly improved matching in the electrophoretic mobilities of single-base extension DNA fragments over the previously described set C10F, C10G, C10T, and C10R and is the best available for sequencing.

DNA↗
PubMed1997

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Buffers↗
PubMed1997

Temperature dependence of the Qy resonance Raman spectra of bacteriochlorophylls, the primary electron donor, and bacteriopheophytins in the bacterial photosynthetic reaction center.

Qy-excited resonance Raman spectra of the accessory bacteriochlorophylls (B), the bacteriopheophytins (H), and the primary electron donor (P) in the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides have been obtained at 95 and 278 K. Frequency and intensity differences are observed in the low-frequency region of the P vibrational spectrum when the sample is cooled from 278 to 95 K. The B and H spectra exhibit minimal changes of frequencies and relative intensities as a function of temperature. The mode patterns in the Raman spectra of B and H differ very little from Raman spectra of the chromophores in vitro. The Raman scattering cross sections of B and H are 6-7 times larger than those for analogous modes of P at 278 K. The cross sections of B and of H are 3-4 times larger at 95 K than at 278 K, while the cross sections of P are approximately constant with temperature. The temperature dependence of the Raman cross sections for B and H suggests that pure dephasing arising from coupling to low-frequency solvent/protein modes is important in the damping of their excited states. The weak Raman cross sections of the special pair suggest that the excited state of P is damped by very rapid (<<30 fs) electronic relaxation processes. These resonance Raman spectra provide information for developing multimode vibronic models of the excited-state structure and dynamics of the chromophores in the RC.

Bacteriochlorophylls↗
PubMed1997

Resonance Raman examination of the wavelength regulation mechanism in human visual pigments.

Resonance Raman spectra of recombinant human green and red cone pigments have been obtained to examine the molecular mechanism of color recognition by visual pigments. Spectra were acquired using a 77 K resonance Raman microprobe or preresonance Raman spectroscopy. The vibrational bands were assigned by comparison to the spectra of bovine rhodopsin and model compounds. The C=NH stretching frequencies of rhodopsin, the green cone pigment, and the red cone pigment in H2O (D2O) are found at 1656 (1623), 1640 (1618), and 1644 cm(-1), respectively. Together with previous resonance Raman studies on iodopsin [Lin, S. W., Imamoto, Y., Fukada, Y., Shichida, Y., Yoshizawa, T., & Mathies, R. A. (1994) Biochemistry 33, 2151-2160], these values suggest that red and green pigments have very similar Schiff base environments, while the Schiff base group in rhodopsin is more strongly hydrogen-bonded to its protein environment. The absence of significant frequency and intensity differences of modes in the fingerprint and the hydrogen out-of-plane wagging regions for all these pigments does not support the hypothesis that local chromophore interactions with charged protein residues and/or chromophore planarization are crucial for the absorption differences among these pigments. However, our data are consistent with the idea that the Schiff base group in blue visual pigments is stabilized by protein and water dipoles and that the removal of this dipolar field shifts the absorption maximum from blue to green. A further red shift of the lambda(max) from the green to the red pigment is successfully modeled by the addition of hydroxyl-bearing amino acids (Ser164, Tyr261, and Thr269) close to the ionone ring that lower the transition energy by interacting with the change of dipole moment of the chromophore upon excitation. The increased hydrogen bonding of the protonated Schiff base group in rhodopsin is predicted to account for the 30 nm blue shift of its absorption maximum compared to that of the green pigment.

Animals↗
PubMed1997

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.

Benzothiazoles↗
PubMed1997

Microsatellite-based cancer detection using capillary array electrophoresis and energy-transfer fluorescent primers.

The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.

DNA, Neoplasm↗
PubMed1997

Energy-transfer fluorescent reagents for DNA analyses.

Fluorescence resonance energy transfer has facilitated the development of a new class of high-performance fluorescent labeling reagents for multiplex analyses of nucleic acids. The enhanced emission of energy transfer (ET) primers has provided a decadic improvement in the performance of automated DNA sequencers. The emission spectral purity of ET primers permits the development of robust multiplex diagnostic methods for the detection of PCR products. High affinity bifunctional intercalation reagents containing ET-coupled dyes are also being used for high-performance multiplex assays of double-stranded DNA when noncovalent labeling is preferred.

Base Sequence↗