The use of pulsed fields in a standard manual sequencing set-up results in the separation of over 2000 bases on a single gel. However visual reading of the sequence from a film is not possible for more than about 800 - 900 bases under the best conditions. The use of image reconstruction and analysis techniques allows the reading of the M13 mp18 sequence up to about 1600 - 1700 bases, and individual bands can be identified above 2000 bases.

The two-color peak-height encoded DNA sequencing technique was evaluated with six clones taken from the malaria genome. This technique produced a sequencing accuracy of at least 97.5%. Capillary gel electrophoresis, using a modest voltage of 200 V/cm, showed a three- fold increase in speed and higher efficiency compared to conventional slab gel technique.

A laser-excited, confocal-fluorescence scanner is used for high-sensitivity, on-column detection of electrophoresis performed using an array of capillaries. The capabilities of this method and apparatus are illustrated by application to high-speed, high-throughput DNA sequencing. Sanger DNA sequencing fragments are separated on an array of capillaries and then distinguished by using a binary coding scheme that employs only two different fluorescently labeled dye primers to identify four sets of fragments. DNA sequencing results are presented using a 25-capillary array. This apparatus has the capability of sequencing DNA at a rate of approximately 25,000 bases/hour.

Since its development, capillary gel electrophoresis has demonstrated the ability to separate DNA sequencing reactions at speeds roughly 25 times as great as conventional slab gel electrophoresis. These increased speeds are the result of using the more efficient dissipation of Joule heating by capillaries. However, to date there have been no studies which quantitate the advantages of disadvantages in operating these gels at high electric field strength. This work addresses this question by investigating the band-broadening of DNA sequencing reactions as they are separated through a fixed distance of gel at field strengths ranging from 50 V/cm to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in diffusional broadening. However, at sufficiently high electric field strengths, the bands begin to broaden again under the influence of an increasing thermal gradient across the diameter of the capillary. The result is an optimum electric field strength in the intermediate range of 100 - 250 V/cm depending on the length of fragments being separated. The relative importance of diffusion and thermal gradients are discussed and used to generate an equation that models the observed band broadening of DNA in capillary gel electrophoresis (CGE).

Stable isotopes can be used as alternatives to radioisotopes or fluorescent DNA labels. After the labeled DNA has been banded on electrophoresis gels the stable isotopes may be located by scanning the gel with resonance ionization spectroscopy. Methods for synthesizing appropriate labels and for detecting isotopes of iron and tin directly on polyacrylamide gels are described.

We present results of STM and AFM investigations of coated and uncoated samples containing plasmid DNA, chemically modified DNA and tobacco mosaic virus. These specimens were adsorbed by a variety of methods onto low Miller index gold single crystals, co-evaporated films, and mica substrates. Some of the samples were prepared and transferred into an ultrahigh vacuum chamber for further treatment and analysis by Auger electron spectroscopy (AES) and electron spectroscopy for chemical analysis (ESCA or XPS) in an effort to investigate various methods for depositing chemically modified DNA onto gold and mica substrates. These results will be discussed in the context of corroborating STM and AFM image results with the established techniques of AES and ESCA. It is potentially beneficial to make certain chemical modifications to the surfaces and the DNA for two purposes: to aid in adsorption of the molecule to the substrate and to provide a label for the electron spectroscopy verification studies. We hope to enhance the molecule-substrate interactions in such a way as to make the imaging of the biomolecules such as DNA more reproducible and less prone to artifacts.

Alkanethiols self-assemble into monolayers on gold surfaces. It has been shown that gold surfaces derivatized with two-carbon, bifunctional alkanethiols differentially adsorb DNA. Gold surfaces modified with either 2-(N,N-dimethylamino)ethanethiol or 2-aminoethanethiol immobilized DNA at solution pH's where the amino end groups are protonated. The cationic layer holds the DNA in place by ion-pairing with the negatively-charged phosphate groups on the DNA backbone. This ion-pairing is sufficiently strong to resist changes in the DNA's location and conformation induced by the scanning tunneling microscope (STM) tip. With these chemically modified surfaces, the reliable and reproducible imaging of DNA is possible. When the length of the alkane spacer is increased to eleven carbons, the observed affinities for radiolabeled DNA are comparable to that observed for the two carbon spacer. However, clearly resolved STM images of DNA immobilized on 11-(N,N'-dimethylamino)- undecanethiol-modified gold have not been obtainable. We hypothesize that images of immobilized DNA are not observed because of the interaction of the scanning probe with the self-assembled alkanethiol monolayer.

Scanning tunneling microscopy, although capable of yielding very high resolution on periodic structures, very often provides only moderate resolution on singular features. Our work aims at the use of laser light to improve the identification of individual molecules. We report on scanning tunneling microscopy measurements performed on dye molecules dissolved in a liquid crystal and adsorbed onto highly oriented pyrolytic graphite. Either localized perturbations of the liquid crystal structure with the size of single molecules or more or less extended ordered domains of well resolved dye molecules were reproducibly imaged for several dyes. To study light-induced resonant effects the influence of non-resonant absorption leading to thermal expansion of tip and sample has to be suppressed. Therefore, an electro- optical system was realized using an Ar^PLU- and a dye laser of different wavelengths power-modulated with a relative phase shift of 180 degree(s). Preliminary results obtained with this setup are presented documenting the efficiency of the compensation.

A simple calculation shows that the information contained in the base sequence of the human genome could be recorded onto less than two compact discs. To read amounts of information comparable in size to the human genome, scanning probes are used routinely in both biology (i.e., living systems) and technology. The atomic force microscope (AFM) is a scanning probe that is now capable of imaging DNA routinely and reproducibly. The minimum size of structures seen reproducibly along DNA strands with the AFM is presently 2 to 3 nm, which is an order of magnitude less resolution than would be required to sequence DNA. At present, the AFM shows great potential for high-resolution mapping of DNA but is not capable of sequencing DNA without further improvements.

We are developing methods for random and site-specific labeling of individual DNA molecules to facilitate manipulation of fragments excised in the atomic force microscope (AFM) and for localization of specific DNA domains, such as protein binding sites and origins of replication. One successful method was to incorporate biotinylated nucleotides at random internal locations or specifically at the ends of linearized DNA molecules in vitro. Following complex formation with 5 nm diameter streptavidin-gold conjugates, chromatographic purification and passive adsorption of the complexes of mica, the biotinylated domains were easily localized in the AFM by virtue of the distinctive size and shape of the streptavidin-gold complex. In many cases unconjugated streptavidin (i.e., lacking gold) was also observed attached to the biotinylated DNA. A second approach to site-specific labeling of DNA for imaging in the AFM was to react DNA with restriction enzymes having sequence-specific binding properties. Like the unconjugated streptavidin-DNA complexes, these enzyme-DNA complexes were visible without attached colloidal gold. Efforts to image DNA labeled in vivo using bromodeoxyuridine (BrdU) and anti-BrdU antibodies are ongoing.

Artificial SFM images of DNA calculated from molecular models show that very sharp tips are needed if the internal features of DNA molecules are to be resolved. The calculations are based on 'envelope image analysis', which takes into account only the geometric effect of a tip surface sliding over a sample surface. These calculations show that: (1) A tip of about 5 angstroms end radius of curvature is needed to profile the major groove of double stranded B- form DNA, and an even sharper tip is needed to profile the minor groove. A tip of 1.5 angstroms radius fully profiles the major and minor grooves, and distinguishes the phosphate groups along the backbone. A tip of 20 angstroms end radius shows only a smooth cylinder with no internal detail. (2) it is possible to distinguish various conformations of double- stranded DNA if the backbone spacing is resolved. This seems to require at least a 5 angstroms tip. (3) The individual bases of single-stranded DNA are not easily distinguishable even if the DNA is in a conformation where the bases lie flat on the substrate surface, and even if a very sharp 1.5 angstroms tip is used.

Techniques have been developed to volatilize intact massive DNA molecules using pulsed laser ablation of thin frozen films of aqueous DNA solutions. Electrophoresis assay of the ablated DNA shows that molecules as massive as approximately 400,000 Da can be ablated intact. It has been possible to obtain time-of-flight mass spectra of ablated multicomponent mixtures of single-stranded DNA with masses up to approximately 18,000 Da (a 60-nucleotide DNA oligomer). The possible application of time-of-flight mass spectrometry to the analysis and readout of DNA sequence mixtures, and the potential thereby to accelerate the Human Genome project, are discussed.

During the past few years, tremendous effort has been put to achieving fast sequencing of DNA due to the potential great applications in biological and medical research. Current successful DNA sequencing methods which were developed by Sanger and Maxam-Gilbert have proven very useful for sequencing small DNA segments. However, these methods of sequencing usually involve labeling fragments of DNA for identification following time- consuming gel electrophoresis. The labeling processes usually involve either radioactive tagging or chromophore tagging to various sizes of DNA segments. The segments of DNA are generated either chemically or enzymatically to represent all possible positions of each of the four nucleotides (A, G, C, and T). Both radioactive methods and fluorescent dye labeling methods for DNA sequencing require the use of the time-consuming gel electrophoresis method. It is naturally desirable to consider a time-of-flight mass spectrometer approach to DNA sequencing since the time needed for separation of DNA segments in a mass spectrometer is a few microseconds instead of a few hours in gel electrophoresis.

Efficient sequencing of the human genome will require the development of new methods that are less expensive and orders of magnitude faster than current technology. Recent advances in laser-based methodology suggest that a mass spectroscopic DNA sequencing technique may surmount present limitations. This contribution will focus on the use of laser vaporization and laser ionization to prepare single stranded DNA for high speed sequencing in the gas phase. As a first step in the implementation of a mass spectroscopic sequencing approach, we have shown that single-stranded DNA molecules having chain lengths of over 1000 nucleotides can be laser vaporized into the gas phase with no discernible strand cleavage. This observation provides the basis for the time-of-flight (TOF) mass spectral-based sequencing experiment that we are developing. To determine the DNA sequence the experiment will be repeated for the four complimentary dideoxy sequencing reactions. The realization of this method would allow a 300 base DNA sequence to be determined in less than one second. At that rate, an instrument based on this technology could potentially generate sequencing data in excess of 25 million bases per day.

A new technology is introduced for developing potentially low cost, high throughput DNA sequence analysis. This approach utilizes novel bioelectronic genosensor devices to rapidly detect hybridization events across a DNA probe array. Detection of DNA probe/target hybridization has been achieved by two electronic methods. The first method utilizes a permittivity chip which interrogates the miniature test fixtures with a low voltage alternating electric field. The second method, which is the emphasis of this paper, utilizes a charge- coupled device (CCD) to detect the hybridization of appropriately tagged (radioisotope, fluorescent, or chemiluminescent labels) target DNA to an array of DNA probes immobilized above the pixels. Such direct electronic-biologic coupling is shown to provide a tenfold sensitivity improvement over conventional lens-based detection systems.

We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.