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P Matsudaira

Publications and source records attributed to P Matsudaira.

At least 19 recordsLinked to original sources
PubMed2001

Shear-induced degradation of linear polyacrylamide solutions during pre-electrophoretic loading.

Electrophoretic channels are filled with a polymer matrix prior to their use in DNA separations. This process, called gel-loading, can be accomplished manually, using syringes, or can be automated through the use of small pumps or vacuum. The injection rate is constrained by the desire to minimize shear-induced degradation of the polymer molecules. Currently, the community lacks quantitative data with which to gauge the range of flow rates that prevent polymer degradation. In this study, measurements of the zero shear rate viscosity of linear polyacrylamide (LPA) solutions are used to determine the LPA molecular weight before and after gel-loading. The results indicate molecular degradation in polymer solutions even when injected at minimal flow rates of 1 microL/min. To correlate these rheological observations of shear-induced degradation with subsequent electrophoretic performance, the degraded solutions were used as sieving matrixes for DNA sequencing analysis. The decreases in electrophoretic resolution and increases in peak widths between sheared and nonsheared LPA solutions are related to the degradation in molecular weight experienced by the polymer solutions.

Calibration↗
PubMed2001

An atomic model of actin filaments cross-linked by fimbrin and its implications for bundle assembly and function.

Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin-fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin-fimbrin bundles. Furthermore, the assembly of the actin-fimbrin arrays suggests coupling between actin polymerization, fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity.

Actin Cytoskeleton↗
PubMed2001

A closed-cycle capillary polymerase chain reaction machine.

A novel thermocycling machine based on a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors is described. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm-i.d., oil-filled capillary using a multielement scattered light detector and active feedback. Dwell times and accelerations can be adjusted independently. As a demonstration of the device, 30 cycles of a 500-base pair product were performed in 23 min with 78% amplification efficiency. This result compares well with previous high-speed thermocyclers. Theoretically, the arrangement can approach a time of 2.5 min for 30 cycle amplifications of a 500-base pair product.

DNA↗
PubMed2000

Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices.

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.

Base Sequence↗
PubMed2000

Eight hundred-base sequencing in a microfabricated electrophoretic device.

The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.

Base Sequence↗
PubMed2000

Microchip electrophoresis: a method for high-speed SNP detection.

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.

DNA Mutational Analysis↗
PubMed2000

Motility powered by supramolecular springs and ratchets.

Not all biological movements are caused by molecular motors sliding along filaments or tubules. Just as springs and ratchets can store or release energy and rectify motion in physical systems, their analogs can perform similar functions in biological systems. The energy of biological springs is derived from hydrolysis of a nucleotide or the binding of a ligand, whereas biological ratchets are powered by Brownian movements of polymerizing filaments. However, the viscous and fluctuating cellular environment and the mechanochemistry of soft biological systems constrain the modes of motion generated and the mechanisms for energy storage, control, and release.

Actins↗
PubMed2000

Brush border myosin I (BBMI): a basally localized transcript in human jejunal enterocytes.

To extend our recent observation that villin mRNA, encoding an apical microvillous protein, is dichotomously localized in the basal region of human enterocytes, we examined the localization of mRNAs for brush border myosin I (BBMI) and intestinal fimbrin (I-fim). In situ hybridization indicated that BBMI mRNA localized to the basal region of human enterocytes, whereas the mRNA for I-fim distributed diffusely. To facilitate study of potential mechanisms of mRNA targeting, we cloned a full-length cDNA for BBMI including its 5'- and 3'-untranslated regions (UTRs). This cDNA shares 86% sequence identity with bovine BBMI and 85% with rat BBMI. Sequence analysis revealed no obvious similarity between the 3'-UTRs of BBMI and villin. This study provides evidence of novel sorting pathways for intestinal microvillous cytoskeletal proteins.

Base Sequence↗
PubMed1999

Scaling structure factor amplitudes in electron cryomicroscopy using X-Ray solution scattering.

The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.

Acrosome↗
PubMed1999

The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 microm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Acrosome↗
PubMed1999

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.

Actins↗
PubMed1999

Two-color multiplexed analysis of eight short tandem repeat loci with an electrophoretic microdevice.

Single-channel microfabricated electrophoretic devices equipped with a dual-wavelength laser-induced fluorescence detection system were used for the fast analysis of an eight-loci, two-color multiplex short tandem repeat (STR) system for human identification. Routine analyses of the eight loci (CSF1PO, TPOX, TH01, vWA and D16S539, D7S820, D13S317, D5S818), requiring four-base resolution, were performed in only 2 min. Specific analyses for a microvariant allele (allele 9.3 of the TH01 locus) demanded single-base resolution and was performed in less than 10 min. The high accuracy of the microdevice for real-world STR sample analyses was demonstrated by comparison with conventional slab-gel electrophoresis. Our results show that a fast multiwavelength multichannel electrophoretic microsystem will be capable of routinely processing thousands of complex STR samples per day.

Color↗
PubMed1999

Recent developments in DNA sequencing by capillary and microdevice electrophoresis.

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine-tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.

Coloring Agents↗
PubMed1999

Microfluidic devices for DNA analysis.

Microfabricated electrophoresis devices allow us to perform short-tandem-repeat genotyping assays in under 2 min and sequence single-stranded DNA in under 15 min. This is 10-100 times faster than standard slab-gel and capillary systems. The microdevice format is the natural extension of 100 years of gradual improvements to electrophoresis but operates in an almost-perfect way, limited only by the sieving medium.

Acrylic Resins↗
PubMed1999

Toward real-world sequencing by microdevice electrophoresis.

We report results using a microdevice for DNA sequencing using samples from chromosome 17, obtained from the Whitehead Institute Center for Genome Research (WICGR) production line. The device had an effective separation distance of 11.5 cm and a lithographically defined injection width of 150 microm. The four-color raw data were processed, base-called by the sequencing software Trout, and compared to the corresponding ABI 377 sequence from WICGR. With a criteria of 99% accuracy, we achieved average continuous reads of 505 bases in 27 min with 3% linear polyacrylamide (LPA) at 150 V/cm, and 460 bases in 22 min with 4% LPA at 200 V/cm at a temperature of 45 degrees C. In the best case, up to 565 bases could be base-called with the same accuracy in <25 min. In some instances, Trout allowed for accurate base-calling down to a resolution R as low as R = 0.35. This may be due in part to the high signal-to-noise ratio of the microdevice. Unlike many results reported on capillary machines, no additional sample cleanup other than ethanol precipitation was required. In addition, DNA fragment biasing (i.e., discrimination against larger fragments) was reduced significantly through the unique sample injection mechanism of the microfabricated device. This led to increased signal strength for long fragments, which is of great importance for the high performance of the microdevice.

Base Sequence↗
PubMed1998

DNA sequencing on microfabricated electrophoretic devices.

We present a model that quantitatively describes the performance of microfabricated electrophoretic devices filled with linear polyacrylamide as replaceable sieving material for single-stranded DNA analyses. The dependence of resolution on various separation parameters such as selectivity, diffusion, injector size, device length, and channel folding was investigated. A previously predicted dependence of longitudinal diffusion coefficient on electric field strength has been verified. We have used this model to develop and optimize microfabricated electrophoretic devices for DNA analyses. For single-color DNA sequencing mixtures, we routinely achieve separations of 400 bases in under 14 min at 200 V/cm, and separation of 350 bases in only 7 min at 400 V/cm, with a minimum resolution of R = 0.5. Our results also indicate reduced fragment biasing and efficient sample stacking for DNA sample loading on microfabricated devices.

Acrylic Resins↗
PubMed1998

An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation.

Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

Actins↗