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Biomedical subjects

Andre Marziali

Publications and source records attributed to Andre Marziali.

10 recordsLinked to original sources
PubMed2006

Multi-nanopore force spectroscopy for DNA analysis.

The need for low-cost DNA sequence detection in clinical applications is driving development of new technologies. We demonstrate a method for detection of mutations in a DNA sequence purely by electronic means, and without need for fluorescent labeling. Our method uses an array of nanopores to perform synchronized single-molecule force spectroscopy measurements over many molecules in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement.

Mutation↗
PubMed2006

Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions.

No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing.

Blood Platelets↗
PubMed2005

Simple, robust methods for high-throughput nanoliter-scale DNA sequencing.

We have developed high-throughput DNA sequencing methods that generate high quality data from reactions as small as 400 nL, providing an approximate order of magnitude reduction in reagent use relative to standard protocols. Sequencing of clones from plasmid, fosmid, and BAC libraries yielded read lengths (PHRED20 bases) of 765 +/- 172 (n = 10,272), 621 +/- 201 (n = 1824), and 647 +/- 189 (n = 568), respectively. Implementation of these procedures at high-throughput genome centers could have a substantial impact on the amount of data that can be generated per unit cost.

Nanotechnology↗
PubMed2005

Novel electrophoresis mechanism based on synchronous alternating drag perturbation.

We present a novel means of transporting molecules in solution by applying a zero-time-average alternating motive force to the molecules, and perturbing the molecular drag coefficient synchronously with the applied force, thus causing a net drift in a direction determined by the phase of the alternating drag perturbation relative to the alternating force. We apply an electrophoretic form of the method to transport and concentrate DNA in a gel, such that all molecules migrate on average away from the nearest electrode and toward a central region. Since an electrode does not occupy this central region, this method presents the possibility of transporting and focusing DNA and other charged molecules in regions free from electrodes and the associated electrochemistry.

DNA↗
PubMed2005

Contaminant-induced current decline in capillary array electrophoresis.

High-throughput capillary array electrophoresis (CAE) instruments for DNA sequencing suffer to varying degrees from read length degradation associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual amounts of large, slow-moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we investigate the creation and expansion of an ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. Slow-moving fragments are analytically and experimentally shown to reduce the ionic concentration of the downstream electrolyte. With injection of large fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region begins to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions, the depletion region expands, the capillary current declines dramatically, and the electrophoresis run yields a short read length or fails completely.

DNA↗
PubMed2004

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Animals↗
PubMed2004

A nanosensor for transmembrane capture and identification of single nucleic Acid molecules.

We have engineered a nanosensor for sequence-specific detection of single nucleic acid molecules across a lipid bilayer. The sensor is composed of a protein channel nanopore (alpha-hemolysin) housing a DNA probe with an avidin anchor at the 5' end and a nucleotide sequence designed to noncovalently bind a specific single-stranded oligonucleotide at the 3' end. The 3' end of the DNA probe is driven to the opposite side of the pore by an applied electric potential, where it can specifically bind to oligonucleotides. Reversal of the applied potential withdraws the probe from the pore, dissociating it from a bound oligonucleotide. The time required for dissociation of the probe-oligonucleotide duplex under this force yields identifying characteristics of the oligonucleotide. We demonstrate transmembrane detection of individual oligonucleotides, discriminate between molecules differing by a single nucleotide, and investigate the relationship between dissociation time and hybridization energy of the probe and analyte molecules. The detection method presented in this article is a candidate for in vivo single-molecule detection and, through parallelization in a synthetic device, for genotyping and global transcription profiling from small samples.

Avidin↗
PubMed2003

Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules.

Nanoscale alpha-hemolysin pores can be used to analyze individual DNA or RNA molecules. Serial examination of hundreds to thousands of molecules per minute is possible using ionic current impedance as the measured property. In a recent report, we showed that a nanopore device coupled with machine learning algorithms could automatically discriminate among the four combinations of Watson-Crick base pairs and their orientations at the ends of individual DNA hairpin molecules. Here we use kinetic analysis to demonstrate that ionic current signatures caused by these hairpin molecules depend on the number of hydrogen bonds within the terminal base pair, stacking between the terminal base pair and its nearest neighbor, and 5' versus 3' orientation of the terminal bases independent of their nearest neighbors. This report constitutes evidence that single Watson-Crick base pairs can be identified within individual unmodified DNA hairpin molecules based on their dynamic behavior in a nanoscale pore.

Algorithms↗
PubMed2002

Evaluation of nanopores as candidates for electronic analyte detection.

In an effort to increase throughput and decrease the cost of electrophoretic separation of DNA and proteins, various groups are developing highly parallel, miniaturized separation devices based on capillaries etched into silicon, glass or plastic substrates. To date, these miniaturized devices have relied on optical detectors, thus placing a lower limit on instrument size, and complicating the incorporation of an entire DNA analyzer instrument on a chip. To address this limitation, we are evaluating nanopores as candidate Coulter counters for purely electronic detection of analytes in miniaturized electrophoresis and similar separation devices. To establish feasibility of this detection scheme, we have investigated the detection sensitivity of a nanopore sensor through experiments with the alpha-hemolysin (alpha-HL) ion channel, and through a Monte Carlo (MC) model of polymer capture rate with a cylindrical nanopore under an applied voltage. Experimental and model results are extrapolated to predict the capture rate of synthetic pores operating at higher voltages than presently achievable with protein pores.

Biopolymers↗