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Biomedical subjects

R A Mathies

Publications and source records attributed to R A Mathies.

At least 73 records · Page 4Linked to original sources
PubMed1995

Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy-transfer fluorescent primers.

Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.

Alleles↗
PubMed1994

Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of phi X174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 microns) have been explored. By use of channels with an effective length of only 3.5 cm, separations of phi X174 Hae II DNA fragments from approximately 70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQ alpha alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.

Cellulose↗
PubMed1994

Vibrationally coherent photochemistry in the femtosecond primary event of vision.

Femtosecond pump-probe experiments reveal the impulsive production of photoproduct in the primary event in vision. The retinal chromophore of rhodopsin was excited with a 35-femtosecond pulse at 500 nanometers, and transient changes in absorption were measured with 10-femtosecond probe pulses. At probe wavelengths within the photo-product absorption band, oscillatory features with a period of 550 femtoseconds (60 wavenumbers) were observed whose phase and amplitude demonstrate that they are the result of nonstationary vibrational motion in the ground state of the photoproduct. The observation of coherent vibrational motion of the photoproduct supports the idea that the primary step in vision is a vibrationally coherent process and that the high quantum yield of the cis-->trans isomerization in rhodopsin is a consequence of the extreme speed of the excited-state torsional motion.

Animals↗
PubMed1994

High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes.

Fluorescence-detected capillary electrophoresis separations of phi X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from phi X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. The optimum concentrations of 9AA for the separation of complexes preformed with the dimeric dyes TOTO, EthD, TOTAB, and YOYO were determined to be 100, 1, 1, and 0.5 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1994

What makes red visual pigments red? A resonance Raman microprobe study of retinal chromophore structure in iodopsin.

We have obtained resonance Raman spectra of iodopsin, a red-sensitive (lambda max 571 nm) pigment from chicken cone cells, to investigate the molecular mechanism of the opsin shift in visual pigments. Detergent-solubilized iodopsin samples were examined with a Raman microprobe to obtain spectra from a 77-K photostationary steady-state mixture composed of 11-cis-iodopsin and its 9-cis-isoiodopsin and all-trans-bathoiodopsin photoproducts. The vibrational modes of these species have been assigned by comparison with spectra of the corresponding bovine pigments. The single bond stretching frequencies of the bovine, toad, and chicken pigments are found to exhibit a regular correlation as a function of the pigment absorption maxima that is consistent with the expected effects of increased electron delocalization. The C = NH stretching frequencies of iodopsin and bathoiodopsin are at 1644 and 1638 cm-1, respectively, and shift down to 1621 and 1617 cm-1, respectively, when the nitrogen is deuterated. The C = ND stretching frequencies of the various pigments are found to decrease linearly with increasing absorption maxima, suggesting that at least part of the opsin shift in visual pigments results from weakened electrostatic interaction between the retinal chromophore and its protein counterion. The Raman data are inconsistent with the idea that a charged protein residue is shifted along the chromophore to regulate the opsin shift. Taken together with the mutagenesis and model compound results, these resonance Raman data suggest that the opsin shift between the green and red cone visual pigment arises from two effects. First, Tyr-274 provides increased electrostatic stabilization of the Schiff base-counterion ion pair. Second, the opsin shift is enhanced by the dipolar residues Ser-177 and Thr-282 that interact with the chromophore near the ionone ring to preferentially stabilize the highly dipolar charge distribution of the electronically excited retinal chromophore [Mathies, R., & Stryer, L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2169-2173].

Animals↗
PubMed1993

The first step in vision occurs in femtoseconds: complete blue and red spectral studies.

Femtosecond transient absorption measurements of the cis-trans isomerization of the visual pigment rhodopsin clarify the interpretation of the dynamics of the first step in vision. We present femtosecond time-resolved spectra as well as kinetic measurements at specific wavelengths between 490 and 670 nm using 10-fs probe pulses centered at 500 and 620 nm following a 35-fs pump pulse at 500 nm. The expanded spectral window beyond that available (500-570 nm) in our previous study [Schoenlein, R. W., Peteanu, L. A., Mathies, R. A. & Shank, C. V. (1991) Science 254, 412-415] provides the full differential absorption spectrum of the photoproduct as a function of delay time after photolysis. The high time-resolution data presented here contradict an alternative interpretation of the rhodopsin photochemistry offered by Callender and co-workers [Yan, M., Manor, D., Weng, G., Chao, H., Rothberg, L., Jedju, T. M., Alfano, R. R. & Callender, R. H. (1991) Proc. Natl. Acad. Sci. USA 88, 9809-9812]. Our results confirm that the red-shifted (lambda max approximately 570 nm) photo-product of the isomerization reaction is fully formed within 200 fs. Subsequent changes in the differential spectra between 200 fs and 6 ps are attributed to a combination of dynamic ground-state processes such as intramolecular vibrational energy redistribution, vibrational cooling, and conformational relaxation.

Color Perception↗
PubMed1993

Heterodimeric DNA-binding dyes designed for energy transfer: stability and applications of the DNA complexes.

Spectroscopic studies of the complexes of double-stranded (ds) DNA with the polymethylene-amine linked heterodimers thiazole orange-thiazole blue, thiazole orange-ethidium, and fluorescein-ethidium, in each case show efficient energy transfer from donor to acceptor chromophores (Benson, S.C., Singh, P. and Glazer, A.N. (1993) accompanying manuscript). A quantitative assay of the stability of such complexes during gel electrophoresis is presented. The off-rate of dye from complexes formed at an initial dsDNA bp:dye ratio > or = 10:1 follows strict first-order kinetics. The t0.5 values for the dissociation of a series of related dyes provide a quantitative criterion for the design of DNA-binding fluorophores. Complexes of dsDNA with the monomeric propidium and cyanine dyes, [1-(9-amino-4,7-diazanonyl)-3,8-diamino-6-phenyl-phenanthridinium bromide trihydrobromide] and (N,N'-tetramethyl-1,3-propanediamino)propyl thiazole orange [4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidenyl]-1-(4 ,4,8-trimethyl-4,8-diazanonyl)-quinolinium diiodide], are much more stable than those with their widely used counterparts, ethidium and thiazole orange. Applications of the new dyes in post-staining of gels and in the multiplex detection of DNA restriction fragments are presented.

DNA↗
PubMed1993

Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity.

Thiazole orange homodimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l-2,3- dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA concentration over a range from 0.5 to 100 ng/ml in the presence of 2 x 10(-7) M TOTO or YOYO in 4 mM Tris-acetate/0.1 mM EDTA/50 mM NaCl, pH 8.2 allows sensitive quantitation of double-stranded DNA in a conventional fluorometer. With nucleic acid-dye mixtures in an array of 25-microliters wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as 20 pg of double-stranded DNA can be detected per well. The array scanning method is rapid, has high throughput, and requires small amounts of sample. It also allows quantitation of single-stranded DNA and RNA.

Animals↗
PubMed1993

High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis.

A new method for performing high-speed, high-throughput sizing of DNA has been developed. Samples containing DNA restriction fragments between 70 and 10,000 base pairs in length are electrophoretically separated using capillary arrays in approximately 20 min and detected with high sensitivity using a fluorescence detection system. The separations of phi X174/HaeIII fragments are performed on an array of seven 100-microns i.d., 350-microns o.d. capillaries using replaceable hydroxyethyl-cellulose solutions as the sieving medium. The fragments are fluorescently labeled using ethidium bromide in the running buffer and detected with a laser-excited, confocal-fluorescence scanner. The limit of detection is approximately 1 pg of DNA per band and separations can be detected injecting DNA samples as dilute as 0.1 ng/microliters. Samples were electrokinetically injected from a cassette of microcentrifuge tubes using a format that will facilitate the application of capillary electrophoresis to separations in molecular biology. The feasibility of extending this technique to lower DNA concentrations and to approximately 100 capillary arrays is discussed.

Bacteriophage phi X 174↗
PubMed1993

Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner.

The stable complexes between highly fluorescent, polyfunctional intercalators and dsDNA can be used to detect dsDNA in agarose gels at picogram levels and for multicolor detection of multiplexed dsDNA fragments. Development of additional DNA-binding fluorophores with appropriate spectroscopic properties will expand the range of applications. In principle, the DNA-dye intercalation complexes represent a more sensitive alternative to an established approach to fluorescent labeling and detection of restriction fragments by ligation to single-stranded short oligonucleotides labeled with different fluorochromes, followed by separation on denaturing polyacrylamide gels. The latter technique gives near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases, and has been successfully applied to cosmid mapping. Detection of DNA fragments as intercalation complexes requires that the separations be performed on agarose gels under nondenaturing conditions. Such conditions have been used for extensive mapping of yeast cosmids with postelectrophoresis staining with ethidium bromide. For the patterns on agarose gels, the magnitude of the "error window," which specifies how similar two fragments must be before the corresponding fragments in different digests are paired, was reported to be strongly size dependent. The error window was expanded by a factor of 1.3 for fragments from 400 to 600 bp, 1.2 for fragments from 600 to 800 bp, and 1.1 for fragments from 800 to 1000 bp. Moreover, it was necessary to introduce corrections for systematic differences between size estimates taken from two different gels. For the multiplexing procedure described here, the size estimates for fragments from 600 bp to over 23 kbp were in close agreement with actual sizes as determined from DNA sequence (Table I), and certainly within the error windows given above. The multiplexing procedure should also minimize errors introduced by gel-to-gel variations in mobility, because the standard and unknowns are always run in the same lanes. Kohara et al. established a physical map of almost the entire Escherichia coli chromosome by analysis of a large genomic library. In this case, partial restriction digests were used to generate patterns of fragments and the mapping was performed by agarose gel electrophoresis. The disadvantage of this approach is that fewer fragments are generated. However, this is compensated for by the fact that partial digests reveal the order of the fragments produced and thus greatly increase the amount of information relevant to the question of overlap between different DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzothiazoles↗
PubMed1992

Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.

Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.

Bacteriorhodopsins↗
PubMed1992

DNA sequencing using capillary array electrophoresis.

A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.

Base Sequence↗
PubMed1992

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.

Bacteriorhodopsins↗
PubMed1992

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

Benzoxazoles↗
PubMed1992

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1991

Rapid-flow resonance Raman spectroscopy of bacterial photosynthetic reaction centers.

Rapid-flow resonance Raman vibrational spectra of bacterial photosynthetic reaction centers from the R-26 mutant of Rhodobacter sphaeroides have been obtained by using excitation wavelengths (810-910 nm) resonant with the lowest energy, photochemically active electronic absorption. The technique of shifted excitation Raman difference spectroscopy is used to identify genuine Raman scattering bands in the presence of a large fluorescence background. The comparison of spectra obtained from untreated reaction centers and from reaction centers treated with the oxidant K3Fe(CN)6 demonstrates that resonance enhancement is obtained from the special pair. Relatively strong Raman scattering is observed for special pair vibrations with frequencies of 36, 94, 127, 202, 730, and 898 cm-1; other modes are observed at 71, 337, and 685 cm-1. Qualitative Raman excitation profiles are reported for some of the strong modes, and resonance enhancement is observed to occur throughout the near-IR absorption band of the special pair. These Raman data determine which vibrations are coupled to the optical absorption in the special pair and, thus, probe the nuclear motion that occurs after electronic excitation. Implications for the interpretation of previous hole-burning experiments and for the excited-state dynamics and photochemistry of reaction centers are discussed.

Kinetics↗