MRI/DNA Helyxzion recognizes the market demand and meets the challenge of MRI/DNA imaging diagnosis. Novel imaging protocols are paired with contrast enhancing probes and MAXfel Laser optical excitation to report gene expression and hopefully sequence whole genomes.
By Charles Stevens
Current MRI signals originate from the water molecules in the body. The observed signals rely on the difference of the water environments in the objectives. When such a difference is not obvious, MRI will fail. In this highly demanding areas, such as tumor detections and brain images, MRI can not give any definite information because the differences of water environments are not related to any physiological and pathological conditions. New MRI signaling molecules are urgently needed.
Helyxzion is developing a new set of magnetic signaling molecules for magnetic resonance image (MRI) sequencing diagnostic studies of DNA and large molecule delivery system for genetic therapy.
Helyxzion will develop new sets of signaling molecules for MRI and MRI/DNA Magnetic Resonance Sequencing, Diagnostic and Detection the potential applications of the new signaling molecules include early tumor detections, metabolism images, and body images.
Helyxzion technology is based on the understanding that disease is a genetic process and that a new model for radiology, called molecular imaging, is required for genetic therapies. The methods employed are so sensitive that diseases measured on the genetic level can be detected and corrected before the patient is aware of their symptoms. While conventional medicine of the 20th century treated the effects of disease, genetic medicine in the 21st century, with its complementary DNA sequence imaging techniques, will treat its causes.
DNA imaging (sequencing) answers key clinical questions associated with gene therapies. Through this in vitro medium, clinicians can determine gene defects and in vivo if gene-altering therapies have reached their cellular targets. It reveals the anatomic region where the introduced genes are expressed as well as the onset, magnitude, and duration of expression. Many new molecular therapies are cytostatic, rather than cytotoxic. This means the therapies inhibit cell growth, but they don't kill cells outright, so radiology's mainstay measures of tumor location, diameter, and volume may no longer provide an accurate reading of a patient's condition. The traditional standards for drug dosing are rendered obsolete because genetically targeted therapies may be largely free of side effects. Instead of monitoring physical symptoms, clinicians will use DNA imaging to determine the correct genetic defect and monitor its successful insertion.
MRI/DNA sequencing and 3D imaging would be no more than a fanciful concept without the Helyxzion ANVIL technology to express DNA data graphically and with 3D imaging of the express genome as a digital image of the individual sequenced. Combined with the human genome project completed in 2002, catalogued the sequence of 30,000 genes that make up the human genome. In addition to supplying researchers with the basic ingredients for genetic medicine and imaging, the project created methods and instruments that have accelerated the pace of discovery.
Although DNA sequence imaging may seem exotic, the principles guiding it are familiar to anyone who has practiced nuclear medicine. Many molecular imaging protocols use a radioisotope as a tracer, but even techniques that employ optical or MR imaging modalities rely on some pharmacological means to track the pharmacokinetic properties of the molecular therapies with which they are paired.
DNA imaging differs from conventional techniques, tts designers must overcome cell membrane barriers to deliver the imaging probes to their DNA molecular target minimizing the size and concentration of probe molecules is essential. Typical target concentrations are on pico- or millimolar levels. Because so few molecules are involved, novel strategies must be created to amplify (LASER) the probe's signal to the point that it can be detected.
Whole genome imaging’s best approach is to amplify DNA intracellular, combined it with green fluorescence protein and ex vivo techniques to validate uptake. Researchers have various options when they examine the tissue culture in a petri dish. Fluorescence indicating gene expression ex vivo can be performed before attempting the more costly and technically demanding in vivo imaging techniques.
A radio labeled probe that is selectively phosphorylated in much the same way that fluorodeoxyglucose is phosphorylated by hexokinase. From that starting point, devise strategies to examine DNA in the most straightforward way.
Monocrystalline iron oxide nanoparticles, an MR contrast agent that is a leading candidate for this role. Each particle comprises about 2000 atoms of iron in a crystalline form wrapped with dextrin. When the dextrin is cross-linked, the particles are called CLIONS (cross-linked iron oxide nanoparticles).
Although this contrast-enhancing behavior can be used to improve the efficacy of MRI for cancer diagnosis, the MGH team plans to report gene expression with the aid of the agent. It has developed vectors that position a therapeutic gene and a transferrin gene side by side in a vector. The expression of the transferrin receptor gene product therefore is a surrogate measure for the expression of the therapeutic gene product.
While the MION protocol is an example of a surface receptor encoding, researchers at the California Institute of Technology are developing MRI contrast agents that become activated at an intracellular level. EgadMe is the most advanced agent thus far developed in this class of selectively activated agents, said Dr. Thomas J. Meade, senior research associate in biology. It consists of a chelator that occupies eight of the nine coordination sites on a gadolinium contrast ion. A galactopyranose residue caps off the remaining coordination site on a gadolinium ion. In this water-inaccessible configuration, the contrast agent is "inactive," meaning it does not affect the T1 times of MRI images.
MRI/DNA Imaging Approaches with Laser Optical Enhancement
Laser Optical imaging will also become an important DNA imaging modality, in part because of new activatable fluorescent contrast agents. Laser Optical imaging uses light waves in a manner similar to x-rays at much higher frequencies. Extremely high spatial resolution is possible with Laser optical imaging, but the clinical value of enhanced coherent Laser has been limited to transparent objects or opaque tissue thinner than 150 mm. With a new generation of fluorescent contrast agents, extending its reach into DNA imaging, in certain chemical configurations, these agents have no effect on the viewed fluorescent image The fluorochromes are unbound after specific enzymatic interaction, however, and these encounters cause them to glow. In some cases, the emission of photons boosts the contrast 1000-fold. This enables target detection with near-infrared fluorescence imaging down to the 10-8 molar concentration level.
The Future
What does this all mean for radiology in 2010? New diagnostic procedures and agents will help identify either the genotype or phenotype of abnormalities in vivo, making cancer combined with in vitro DNA sequencing will make comprehensive diagnosis possible. Breast adenocarcinoma, for example, is thought to be at least two different diseases. It is a safe bet that by 2010, radiologists will be determining optimal therapy by using in vivo LMI and in vitro DNA imaging to identify these unique genetic profiles.
Activatable MR agents will play a major role, researchers at Telomolecular Corp. have a FDA approved large molecules delivery system to penetrate cell membranes and to deal with the high mass levels of probes required to produce sufficient signal for DNA imaging.
Laser enhanced MRI imaging will become prominent because it provides benefits beyond the capabilities of other imaging technologies. "This will be the classic case of early diagnosis with DNA imaging, perhaps before morphologic or clinical phenotypic signs of disease can be seen.
Helyxzion predicts that some of today's mainstream applications will appear quaint 10 years from now. At that time, no one will recommend serial scans separated by three-month intervals to monitor the efficacy of chemotherapy based on the size of a tumor. Instead, Helyxzion foresees that a genetic profile of the patiant will be generated and an Individualized therapeutic plans will be formulated based on genetic profiles.
Serendipity makes accurate prediction difficult, and random events interfere with well-intentioned forecasting. "It is clear; however, that the first DNA imaging to obtain FDA approval for clinical use will be with Laser enhanced MRI.
According to Helyxzion many enabling technologies are contributing to DNA imaging research, , Nano-device engineering, improved data processing are making there mark but the ability to 3D image the sequencing data will prove to be by far the most important.
Helyxzion is seeking business partners to strengthen the company, its manufacture ability and to further develop the MRI technology into clinic applications. The expected investment is between two millions and five millions US dollars. Helyxzion will expect intellectual properties and patents in near future.
Other work being done: Apr 18, 2005
Development of Sequencing Technology
Two grafs on the development of sequencing technology from a recent article in
Bioscience Technology.
1) Sequencing technology is "frozen in time", still searching for a breakthrough:
Progress in gene sequencing has arisen more from improved methods than ground-breaking instrumentation. Glenn Schulman, PharmD, marketing manager at 454 Life Sciences (New Haven, CT) points out that gene sequencing technology has become frozen in time circa 2000. “Things pretty much stopped with capillary electrophoresis-based instrumentation,” he says. “There have been incremental improvements, but nothing truly enabling.”
2) Logarithmic Scaling of
454's sequencing-by-synthesis technology (see
image)
454’s progress has been phenomenal since it reported its first results, on about 25 base pairs, in late 2001. Since then scale-up has been logarithmic: 33 kbp in 2002, 2.8 Mbp in 2003, and about 20 million bp today (about the size of a bacterial genome) in a 4.5 hour run. Dr. Schulman sees no end in sight to Moore’s Law-type scaling, which could result in sequencing a whole human genome — 30 Gbp — in a matter of days or hours.
AGOWA GmbH is expanding its range of technological facilities in the area of high-throughput DNA analysis with the implementation of the ABI PRISM® 3730 xl. This latest innovation in DNA analyzers is distinguished by shorter runtimes, longer read lengths, increased throughput and also provides excellent data quality. AGOWA has many years experience and a proven excellent reputation for large-scale sequencing as demonstrated by their participation in large-scale national and international sequencing projects. The sequencing service provided by AGOWA combines their experience and state-of- the-art technology with the aim to rapidly deliver to clients best quality at favourable prices. AGOWA is a competent outsourcing partner for clients in industry and research and offers a broad range of services ranging from DNA libraries, automatic picking and spotting of clones, custom sequencing, bioinformatics down to complete genome analysis.
Liquid-State NMR and Scalar Couplings in Microtesla Magnetic Fields
We obtained nuclear magnetic resonance (NMR) spectra of liquids in fields of a few microtesla, using prepolarization in fields of a few millitesla and detection with a dc superconducting quantum interference device (SQUID). Because the sensitivity of the SQUID is frequency independent, we enhanced both signal-to-noise ratio and spectral resolution by detecting the NMR signal in extremely low magnetic fields, where the NMR lines become very narrow even for grossly inhomogeneous measurement fields. In the absence of chemical shifts, proton-phosphorous scalar (J) couplings have been detected, indicating the presence of specific covalent bonds. This observation opens the possibility for "pure J spectroscopy" as a diagnostic tool for the detection of molecules in low magnetic fields.
Full size imageIBM scientists have achieved a breakthrough in nanoscale magnetic resonance imaging (MRI) by directly detecting the faint magnetic signal from a single electron buried inside a solid sample. This achievement is a major milestone toward creating a microscope that can make three-dimensional images of molecules with atomic resolution. Success in this quest should have major impact on the study of materials -- ranging from proteins and pharmaceuticals to integrated circuits and industrial catalysts -- for which a detailed understanding of the atomic structure is essential. Knowing the exact location of specific atoms within tiny nanoelectronic structures, for example, would enhance designers' insight into their manufacture and performance. The ability to directly image the detailed atomic structure of proteins would aid the development of new drugs.
"Throughout history, the ability to see matter more clearly has always enabled important new discoveries and insights," says Daniel Rugar, manager of nanoscale studies at IBM's Almaden Research Center in San Jose, California. "This new capability should ultimately lead to fundamental advancements in nanotechnology and biology." Rugar leads the team of scientists who for more than a decade have been making pioneering advancements in the nanoscale MRI method called magnetic resonance force microscopy (MRFM). His team has improved MRI sensitivity by some 10 million times compared to the medical MRI devices used to visualize organs in the human body. The improved sensitivity extends MRI into the nanometer realm. (A nanometer is a billionth of a meter, the length spanned by about 5-10 atoms.) IBM Research has a distinguished history in developing microscopes for nanoscale imaging and science. Gerd Binnig and Heinrich Rohrer of IBM's Zurich Research Laboratory received the 1986 Nobel Prize in Physics for their invention of the scanning tunneling microscope, which can image individual atoms on electrically conducting surfaces. Binnig later invented the atomic force microscope (AFM), which used the attraction between a cantilever and surface features on non-conducting surfaces. Scientists at IBM and elsewhere modified and extended the AFM design to image surface forces such as magnetism, friction and electrostatic attraction with nanometer resolution. MRFM combines concepts from both AFM and MRI to allow nanometer resolution of features up to 100 nanometers deep inside a sample. The IBM team of Rugar, John Mamin, Raffi Budakian and Benjamain Chui published its single-electron results in the July 15 issue of the scientific journal Nature. This research is funded in part by the Defense Advanced Research Projects Agency. Technical details The central feature of an MRFM is a microscopic silicon "microcantilever" that looks like a miniature diving board 1,000 times thinner than a human hair. It vibrates at a frequency of about 5,000 times a second, and attached to the cantilever tip is a tiny but powerful magnetic particle. Isolated ("unpaired") electrons and many atomic nuclei behave like tiny bar magnets. These fundamental units of magnetism are often called "spins." Just as two bar magnets can attract or repel each another, the MRFM’s magnetic tip is attracted or repelled by the spins in the sample. By tuning an oscillating high-frequency magnetic field to the natural precession frequency of the spin being imaged, its magnetic orientation flips back and forth as the cantilever vibrates. Although the magnetic force between the magnetic tip and the spin is exceedingly small (less than a millionth of a trillionth of a pound), the cantilever is so sensitive that the flipping of the spin causes a detectable change in the cantilever’s vibration frequency. While medical MRI looks at groups of at least 1 trillion proton spins, the IBM researchers have just detected the much fainter signal of a single electron spin. The researchers also demonstrated rudimentary (one-dimensional) imaging with 25-nanometer resolution, about 40 times better than the best conventional MRI-based microscopes. Rugar's future research is aimed at further improving the sensitivity, resolution and speed of the MRFM technique so it can detect single protons and other nuclei, such as carbon-13, that can be used to reveal molecular structures. (The magnetic signal of a single electron is about 600 times stronger than that of a single proton.) Applying MRFM to protein structures would be particularly far-reaching. The biological activity of a large protein molecule is determined by its intricately folded atomic configuration. But since such a structure is currently impossible to determine directly, scientists must use indirect methods such as the scattering of x-rays by crystallized proteins, or computer simulations. Advanced MRFMs may also be able to serve as detectors of quantum information in future spin-based quantum computers.
Polymerases for Sequencing by Synthesis
Significant enhancements in gene sequencing may be achieved through implementation of analysis instruments at the same dimensional scale as DNA, i.e., nanometers. Nanotechnology has recently provided the necessary tools to create such nanoinstruments and this proposal seeks to utilize these tools to fabricate a high-speed, low-cost gene sequencer. The gene sequencer is based on the nanopore approach and incorporates tunneling current electrodes to sense the individual nucleotides as they transverse the pore.
DNA Sequencing Using Nanopores
This project, as its R21 milestone, will deliver a combination of conical nanopores having read length dimensions slightly less than 1 nm, and nucleobase-modified DNA oligonucleotides, where the passage of the DNA through the nanopore proceeds with a time constant of 10-100 microseconds per nucleotide, and where the ion current through the nanopore, during the time when the DNA is in transit, varies detectably depending on the nucleotide that is in the pore at the time that the current is measured. This nanopore-modified DNA combination will form the core of an extremely inexpensive technology to generate long reads of DNA sequence at the single molecule level. The research will exploit a decade of experience in the Martin laboratory preparing nanopores and engineering their chemical context, and an equal experience in the Benner laboratory working with nucleic acid analogs, polymerases that accept them, and practical applications of the combination. As specific aims, we shall: (a) prepare the nanotubes; (b) attach chemical functionality to the nanotubes; (c) prepare nucleoside triphosphates carrying different sized polyether dendrimers attached at the 5-position (for pyrimidines) and the 7-position (for 7-deazapurines); (d) use these triphosphates to synthesize modified DNA molecules. The nanopores will then be physically characterized to determine their ion transport dynamics, and in conjunction with the modified oligonucleotides, to find a combination that meets the R21 milestone specifications. If this milestone is passed, the next period will be used to develop sequence specific and randomly targeted primers that incorporate DNA, PNA, and tags that exploit an artificial genetic alphabet, and to develop improved processes for generating conical nanopores in a form suitable for large scale application. These will then be targeted against specific sequences extracted from mammalian genomes.
Polymerases for Sequencing by Synthesis
This project, as its R21 milestone, will deliver Taq DNA polymerases that catalyze the template-directed addition of nucleoside triphosphates carrying large fluorescent groups attached to their 3'-ends. The fluorescent groups therefore both terminate transiently the growth of the oligonucleotide chain, and signal the nature of the nucleotide that was last added. These polymerase variants will form the core of a "cheap reagent" approach to the Sequencing by Synthesis (SbS) strategy. Gaining control over polymerase behavior is key for this approach to generate inexpensive genome-quality sequence data. The research will exploit a decade of experience in the Benner laboratory with nucleic acid analogs, polymerases that accept them, and practical application of the combination. The tactics assume that site-directed mutagenesis is generally site-directed damage, and therefore must be followed by directed evolution to obtain polymerase-substrate combinations that meet specifications. Here, directed evolution will be used to restore catalytic power and fidelity in polymerases that have been engineered to accept fluorescent tags. We shall: (a) synthesize nucleoside triphosphates that have fluorescent blocking groups; (b) use a directed evolution system in water-in-oil emulsions to select polymerases that accept the triphosphates efficiently and faithfully; (c) obtain polymerases to incorporate these to within 10% the catalytic activity of native polymerases, and with specificity to better than one part in 10,000. The next phase of the project will be to develop a working prototype for a multiplexed sequencing-by-synthesis device using these polymerases. The Aims of that phase will be to: (d) optimize the fluorescent compound-cleavage chemistry-polymerase combination; (e) use an artificially expanded genetic information system (AEGIS), the artificial alphabet invented in the Benner group, to bin primer-template combinations for parallel sequencing; and (f) exploit 2D gels to develop an architecture for a prototype parallel sequencing instrument based on the technologies developed in Aims a-c.
Bead-based Polony Sequencing
The goals of this project are to develop a robust sequencing by synthesis methodology for de novo and resequencing applications using the bead-based polony technology. Our overall R & D focus is to address key aspects of the technology that need to be refined to enable robust, high quality polony sequencing. Our experience in large-scale genome sequencing will serve well to ensure that the key issues involved in optimizing the technology against current industry standards, data processing, management, and analysis are effectively addressed in a time- and cost-efficient manner. The specific aims are to:
Develop effective procedures for production of paired-end PCR libraries with virtual insert sizes (distance between read pairs) in the range of 2 to 50 kilobases.
Develop methods for effective solid-phase template amplification on derivatized microspheres and for enrichment of beads containing amplified templates.
Develop methods for robust array preparation.
Develop procedures for fluorescent in situ sequencing by synthesis.
Develop an integrated data acquisition system including fluorescence microscope, automated stage, flow cell, fluidics system and control software.
Develop data management and assembly software.
Develop functional reversible chain terminators.
Develop modified enzymes capable of efficiently incorporating reversible terminators.
http://72.14.203.104/search?q=cache:SLNqxGPANwIJ:www.wiley-vch.de/books/biotech/pdf/v05b_midd.pdf+sequencing+technology&hl=en&gl=us&ct=clnk&cd=32http://www.spaceref.com/news/viewpr.html?pid=14018http://www.genome.gov/12513162 more info…
New Technology Will Speed Genome Sequencing
CAMBRIDGE, Mass. -- Almost 150 different genomes have been sequenced to date, including the human genome. But sequencing needs are growing faster than ever: In March 2003, the Bush administration announced it will spend $1 billion over five years to increase forensic analysis of DNA, including a backlog of up to 300,000 samples. And the success of the growing field of genomic medicine, which promises to deliver better therapies and diagnostics, depends on faster sequencing technology.
This fall, researchers at Whitehead Institute will test new technology that could aid these and other endeavors. The BioMEMS 768 Sequencer can sequence the entire human genome in only one year, processing up to 7 million DNA letters a day, about seven times faster than its nearest rival. Scientists began working on the project in 1999 with a $7 million National Human Genome Research Institute grant. The technology eventually will help scientists quickly determine the exact genetic sequence of the DNA of many different organisms, and could lead to faster forensic analysis of DNA gathered in criminal cases.
The heart of the new BioMEMs machine is a large glass chip etched with tiny microchannels called "lanes." It tests 384 lanes of DNA at a time, four times more than existing capillary sequencers. Each lane can accommodate longer strands of DNA: about 850 bases (the nucleic acids found in DNA, abbreviated by the letters A, C, T or G), compared to the current 550 bases per lane.
It takes about 45 minutes to read the DNA from one of the BioMEMS' 768 lanes. The machine has two chips; one is prepared as the other is sequenced, so that the machine is sequencing at all times. The new sequencer saves not just capital costs, the developers say, but day-to-day expenses as well.
"It's not only the cost of the machine, but the cost of the materials it uses," says Brian McKenna, a senior software engineer at Whitehead Institute. The goal, he says, is to use the same amount of consumables -- liquid, chemicals, and other materials used to prepare the DNA -- as existing sequencing machines. BioMEMS also uses a DNA loading process that eventually will need only 1 percent of a typical DNA sample.
While developed at Whitehead, the machine is being commercialized by network biosystems, a company in Woburn, Mass., started in 2001 by Whitehead Member Paul Matsudaira, BioMEMS Labs Director Dan Ehrlich and research scientist Lance Koutny. Shimadzu Biotech in Japan will manufacture the sequencer.
DNA sequencing
How to determine the sequence of bases in a DNA molecule.
DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.
The most commonly used method of sequencing DNA - the dideoxy or chain termination method - was developed by Fred Sanger in 1977 (for which he won his second Nobel prize). The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction.
Key principles:
A DNA molecule carries information in the form of four chemical groups or bases, represented by the letters A, C, G and T. The order of bases on a DNA strand is the DNA sequence.
Most DNA sequencing is carried out using the chain termination method. This involves the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues.
The chain termination method produces a set of DNA molecules differing in length by one nucleotide. The last base in each molecule can be identified by way of a unique label. Separation of these DNA molecules according to size places them in the correct order to read off the sequence.
How does it work? The DNA to be sequenced is provided in single-stranded form. This acts as a template upon which a new DNA strand is synthesized. DNA synthesis requires a supply of the four nucleotides (the building blocks of DNA), the enzyme DNA polymerase and a primer (a short sequence annealed to the template which initiates the new DNA strand). The nucleotides added to the growing DNA strand are complementary to those in the template strand.
Sequencing is achieved by including in each reaction a nucleotide analogue that cannot be extended and thus acts as a chain terminator. Four reactions are set up, each containing the same template and primer but a chain terminator specific for A, C, G or T. Because only a small amount of the chain terminator is included, incorporation into the new DNA strand is a random event. Each reaction therefore generates a collection of fragments, but every DNA strand will end at the same type of base (A, C, G or T).
The primers or nucleotides included in each of the four reactions contain different fluorescent labels allowing DNA strands terminating at each of the four bases to be identified. The reaction products are then mixed and separated by gel electrophoresis, which separates DNA molecules according to size even if they differ in length by only a single nucleotide. As the DNA strands pass a specific point, the fluorescent signal is detected and the base identified. The whole process can be extensively automated.
How is it used?The most obvious application of DNA sequencing technology is the accurate sequencing of genes and genomes. Only about 5-800 bases can be sequenced in one experiment so larger DNA molecules, including whole genomes, must be broken into smaller fragments before sequencing and then reassembled by searching for overlaps. Accuracy is achieved by sequencing each template several times.
Lower-fidelity single-pass sequencing is useful for the rapid accumulation of sequence data at the expense of some accuracy. Another application of DNA sequencing technology is resequencing the same DNA molecule over and over. This is necessary, for example, in the typing of single nucleotide polymorphisms.
