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R A Mathies

Publications and source records attributed to R A Mathies.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1996

Retinal analog study of the role of steric interactions in the excited state isomerization dynamics of rhodopsin.

The role of intramolecular steric interactions in the isomerization of the 11-cis-retinal chromophore in the photoreceptor protein rhodopsin is examined with resonance Raman and CD spectroscopy combined with quantum yield experiments. The resonance Raman spectra and CD spectra of 13-demethylrhodopsin indicate that its chromophore, an analog in which the nonbonded interaction between the 10-H and the 13-CH3 groups is removed, is less distorted in the C10...C13 region than the native chromophore. The reduced torsional and hydrogen-out-of-plane resonance Raman intensities further indicate that the excited state potential energy surface has a much shallower slope along the isomerization coordinate. This is consistent with the decrease in quantum yield from 0.67 in rhodopsin to 0.47 in 13-demethylrhodopsin. The resonance Raman intensities show that the steric twist is reintroduced by addition of a methyl group at the C10 position. However, the quantum yield of 10-methyl-13-demethylrhodopsin is found to be only 0.35. This is attributed to nonisomorphous protein-analog interactions. The nonbonded interaction between the 10-hydrogen and the 13-methyl group in 11-cis-retinal makes this isomer particularly effective as the light-sensing chromophore in all visual pigments.

Animals↗
PubMed1996

Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome.

Resonance Raman vibrational spectra of the Pr, lumi-R, and Pfr forms of phytochrome have been obtained using low-temperature trapping and room temperature flow techniques in conjunction with shifted-excitation Raman difference spectroscopy (SERDS). The Pr to lumi-R photoconversion exhibits a thermal barrier and is completely blocked at 30 K, indicating that thermally assisted protein relaxation is necessary for the primary photochemistry. When Pr is converted to lumi-R, new bands appear in the C = C and C = N stretching regions at 1651, 1636, 1590, and 1569 cm-1, indicating that a significant structural change of the chromophore has occurred. The photoconversion also results in an 18 cm-1 decrease in the N-H rocking band in lumi-R. Normal mode calculations correlate this frequency drop with a change in the geometry of the C15 methine bridge of the phytochromobilin chromophore. Additionally, a C = N stretching mode marker band shifts from 1576 cm-1 in Pr to 1569 cm-1 in lumi-R and to 1552 cm-1 in Pfr. Normal mode calculations show that the frequency drop of this band in the lumi-R-->Pfr interconversion is an indication of a C14-C15 syn-->anti conformational change. Moderately intense hydrogen out-of-plane modes that occur at 805 cm-1 in Pr shift to 829 and 847 cm-1 upon photoconversion to lumi-R and are replaced by a very intense mode at 814 cm-1 in Pfr. These observations indicate that the C and D rings of the chromophore in Pr and lumi-R are moderately planar but that they become highly distorted in Pfr. This information suggests that the primary photochemistry in phytochrome is a Z-->E isomerization of the C15 = C16 bond of Pr giving lumi-R. This is followed by a thermal syn-->anti C14-C15 conformational relaxation to form Pfr. A four-state model is presented to explain the chromophore structural changes in Pr, lumi-R, and Pfr that uses hydrogen bonding to the surrounding protein to stabilize the high-energy Pfr C15 = C16, C14-C15, E,anti chromophore structure. This implicates an anchor and release mechanism between the chromophore and protein that might lead to altered biological signaling in the plant.

Hydrogen Bonding↗
PubMed1996

Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers.

Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses (Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nature Med. 2, 246-249). We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-(epsilon-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CYA; epsilon M488nm 142,000 M-1 cm-1) at the 5' -end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; epsilon M488nm 60,000 M-1 cm-1) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capilary sequencer with 100% accuracy in the first 500 bases.

Carbocyanines↗
PubMed1996

Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.

DNA↗
PubMed1996

Energy transfer primers with 5- or 6-carboxyrhodamine-6G as acceptor chromophores.

Energy-transfer (ET) fluorescent primers for DNA sequencing and multiplex genetic analysis (Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351) are named according to the convention D-N-A, where D is the donor, N is the number of bases between the donor and the acceptor, and A is the acceptor. Thus, a primer that carries 6-carboxyfluorescein (FAM) at the 5'-end and 6-carboxy-4', 5'-dichloro-2',7'-dimethoxyfluorescein (JOE) attached to a modified thymidine 10 bases away is designated F10J. We describe here new ET primers, with 5- or 6-carboxyrhodamine-6G (G5 or G6) as acceptors (with FAM as the donor) in place of JOE, with improved match in the electrophoretic mobilities of the DNA fragments extended from the ET dye-labeled primers, and less overlap in the fluorescence emission of the various labeled DNA fragments. This reduced spectral overlap is most likely due to the narrower emission from G5 or G6 in F10G compared to that from JOE in F10J. With single-stranded M13mp18 DNA as the template, a typical run with F10G6 and three other ET primers on a capillary sequencer provided DNA sequences with 99% accuracy in the first 620 bases.

Base Sequence↗
PubMed1996

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.

Base Sequence↗
PubMed1996

High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers.

Short tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5' end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). M13 A-track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high-resolution, high-speed and high-throughput STR typing of single-stranded DNA fragments using capillary array electrophoresis.

Bacteriophage M13↗
PubMed1996

DNA sequencing using a four-color confocal fluorescence capillary array scanner.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

Base Sequence↗
PubMed1995

Ultra-high-speed DNA sequencing using capillary electrophoresis chips.

DNA sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to approximately 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA sequencing with 97% accuracy and single-base resolution to approximately 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA sequencing using capillary array electrophoresis chips.

Base Sequence↗
PubMed1995

Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis.

We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5' end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (448 nm) and fluorescence emission maxima of 525, 555, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and acceptors is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and mobilities of the single base extension DNA fragments generated with primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.

Base Sequence↗
PubMed1995

Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.

Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Bacterial Proteins↗
PubMed1995

Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis.

A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative T06 as they passed through the approximately 2 micrometer diameter focused laser beam. Amplified photoelectron pulses from the photomultiplier are grouped into bins of 360-450 micros in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. Statistical analyses showed that (i) the number of M13 molecules counted versus concentration was linear with slope = 1, (ii) the average burst duration was consistent with the expected transit time of a single molecule through the laser beam, and (iii) the number of detected molecules was consistent with single molecule detection. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The autocorrelation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of approximately 100 DNA molecules/band or better.

DNA↗
PubMed1995

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Bacteriophage M13↗
PubMed1995

Near-infrared resonance Raman spectra of Chloroflexus aurantiacus photosynthetic reaction centers.

Resonance Raman spectra of the photosynthetic reaction center isolated from the green bacterium Chloroflexus aurantiacus have been obtained with excitation in the near-infrared absorption bands of the special pair (P) and the accessory bacteriochlorophyll (B) using shifted-excitation Raman difference spectroscopy (SERDS). These spectra are compared with the previously reported Raman spectra of P and B in reaction centers from the purple bacterium Rhodobacter sphaeroides. The spectra of P and B from the two species are nearly identical. Common and distinctive attributes of these spectra include enhanced low-frequency (30-200 cm-1) modes in P and the absence of strong Raman activity in modes higher than 1200 cm-1 in both P and B. Also, the absolute scattering cross sections with excitation in the P band are unusually weak in both reaction centers, indicating that their excited states are rapidly vibronically dephased. The striking similarities between the P and B spectra in reaction centers from two very different bacterial species suggest that the common nuclear and electronic dynamics identified here are characteristic of photosynthetic reaction centers.

Bacteria↗