Human Genome 1993 Program Report: Mapping Instrumentation
Date Published: March 1994
Human Genome Management Information System
Oak Ridge National Laboratory
1060 Commerce Park, MS 6480
Oak Ridge, TN 37830
423-576-6669, Fax: 423-574-9888
Internet: bkq@ornl.gov
This group is involved in research in two major fields, bacilli genetics and genetic engineering. We have recently begun exploring electrotransformation (ET) as a method for introducing DNA into industrial bacilli. In these projects physics and biology researchers from different St. Petersburg institutes gathered together in ECOGENE, a science technology company.
We are attempting to create a system of highly efficient but gentle methods for increasing cell-membrane permeability and for introducing biomolecules (DNA, protein) into cells via an electric field. These methods appear to have a number of advantages over traditional techniques in that the metal electrodes do not come into contact with the cell suspension; an extremely high intensity of the electric field (400 kV/cm or more) can be achieved with pulse duration of 10 ns and more; and cell survival, electrostimulation efficiency of the microbiological processes, and ET with biomolecules are increased.
The project is based on experiments revealing the unexpected roles of electric field intensity for cell-wall permeability and the dependence of pulse shape on ET efficiency. These observations became apparent through use of a specially constructed apparatus in which electric pulse parameters were independent of cell suspension and pulse shapes could vary.
This project consists of (1) development of electronic equipment and (2) a theoretical study of the biophysics process. We are developing techniques for introducing very large DNA molecules into Escherichia coli cells that cannot be efficiently transformed by the classical method of electroporation. Our immediate goals are the following:
Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow 117984, Russia
Intracellular flow karyotyping appears to be a feasible and beneficial method for analyzing karyotype aberrations from individual cells using flow cytogenetics. This technology might be especially useful for various studies of karyotype instability and tumorigenesis.
Groups headed by Scott Cram (Los Alamos National Laboratory) and Andrei Poletaev (Russian Academy of Sciences) are collaborating to achieve the following six goals. The Russian group is carrying out research described in all six goals; American investigators will concentrate on the last three.
1.Optimize the technology of hydrodynamic destruction of mitotic cells by capillary-flow high-gradient devices.
First, we will establish the main principles of intracellular analysis technology and build two instruments, one for general study and the other for making improvements to the method. Second, we will experiment with human cells and improve equipment and methods. Last, we intend to adapt the new technology for modern serial flow cytometers.
Human Genome Center and (1)Material Science Division; Lawrence Berkeley Laboratory; Berkeley, CA 94720
Small DNA fragments of known length were made using the polymerase chain reaction. These frag-ments had biotin molecules (vitamin H) covalently attached to each end and were then labeled with streptavidin. This tetrameric complex was expected to bind up to four DNA molecules via their attached biotin molecules. The DNA was then imaged with atomic force microscopy (AFM). As expected theoretically, images revealed the protein at the end of the DNA strands as well as the presence of dimers, trimers, and tetramers of DNA bound to a single protein. Imaging time was about 1 min.
With these results, we believe we have shown that AFM does have sufficient resolution to map DNA. In its simplest form, mapping involves measuring the physical distance between two points of DNA. In this experiment we have demonstrated the ability of AFM to perform this task by attaching a large protein marker to genetically engineered pieces of human DNA and using AFM to locate the marker and measure the known length from the protein to the other end of the DNA.
The purpose of this project is to develop means and methods for flow karyotyping and chromosome sorting. Analytical flow karyotyping is being applied to a variety of areas related to genomic research and medical diagnostics. Chromosomes purified by flow sorting are used for the production of clones or DNA libraries amplified by polymerase chain reaction (PCR).
The project is a continuation of the principal investigators' work at Lawrence Livermore National Laboratory. We have extensively explored chromosome analysis by flow cytometry and have improved techniques of chromosome preparation and staining. We have built flow instruments that accurately measure and sort chromosomes with high efficiency. We have produced software that facilitates analysis and comparison of human flow karyotypes and optimizes sort purity and throughput. As a result of these developments, quantitative DNA measurement of human chromosomes from clinical peripheral blood cultures and established cell lines has become a straightforward and reproducible technique that can be applied to a variety of genetic studies. Examples are the description of normal chromosome heteromorphism, quantification of deletion size in contiguous gene syndromes, and routine monitoring of somatic cell hybrids. Our developments in high-speed sorting technology have led to chromosome-enriched libraries (e.g., the DOE gene library project). Our techniques have been adopted by other laboratories, and the instrumentation, which has been licensed to industry, will soon be available commercially.
This work should increase the availability of this technique to the genetics and genomics research community. Experiments will provide quantitative information on normal and abnormal chromosomes, understanding of interactions of DNA-binding dyes and chromatin, deletion maps of particular chromosome regions to facilitate directed disease-gene mapping, improved purification of chromosome types, and simplification of flow technology for export to other research institutions. In addition, techniques will be developed for mapping probes to chromosomes sorted onto filters, producing DNA or RNA sequence libraries by PCR amplification of small numbers of sorted chromosomes or cells, and identifying and handling single bacteria carrying transfected sequences.
Technology Development for Large-Scale Physical Mapping
Advanced Flow Cytometry Technique Development
DNA Separation by Pulsed-Field Capillary Electrophoresis
Image Acquisition and Analysis
Automated Methods for Large-Scale Physical Mapping
Robotics and Automation
+7-095/135-9824, Fax: -1405 or /938-2187, Internet: polet@imb.msk.su
(1)St. Petersburg Institute of Nuclear Physics and (2)Institute of Cytology; Russian Academy of Sciences; St. Petersburg, Russia
(3)Institute of Molecular Biology; Russian Academy of Sciences
(4)Physico-Technical Institute; Moscow, Russia
2.Develop alternative methods (particularly ultrasonic disintegration) of cell membrane destruction.
3.Improve methods for preparing human cells for analysis while maintaining stable chromosome staining inside the cells.
4.Adapt the method for modern serial flow-cytometer systems.
5.Develop new algorithms and computer programs for data interpretation.
6.Conduct pilot research using different human cell line models to investigate the method's parameters.Atomic Force Microscopy of Biochemically Tagged DNA
Matthew N. Murray, Helen Hansma,(2) D. Frank Ogletree,(1) William F. Kolbe, Sylvia Spengler, Cassandra Smith,(3) Charles Cantor,(3) and Miguel Salmeron(1)
Salmeron: 510/486-6230, Fax: -4995, Internet: salmeron@lbl.gov
(2)Department of Physics; University of California; Santa Barbara, CA 93106
(3)Center for Advanced Research in Biotechnology; Boston University; Boston, MA 02215Flow Karyotyping and Flow Instrumentation Development
Ger van den Engh and Barbara Trask
Department of Molecular Biotechnology; School of Medicine; University of Washington; Seattle, WA 98195
206/685-7345, Fax: -7301, Internet: engh@fishnet.mbt.washington.eduProjects Continuing into FY 1993
Tony J. Beugelsdijk, Patricia A. Medvick, Robert M. Hollen, and Randy S. Roberts
Los Alamos National Laboratory; Los Alamos, NM 87545
505/667-3169, Fax: /665-3911
James H. Jett, John C. Martin, and Mark E. Wilder
Center for Human Genome Studies; Life Sciences Division; Los Alamos National Laboratory; Los Alamos, NM 87545
505/667-3843, Fax: /665-3024, Internet: jett@flovax.lanl.gov
Barry L. Karger
Barnett Institute; Northeastern University; Boston, MA 02115
617/437-2867, Fax: -2855
William F. Kolbe, Joseph E. Katz, and Joseph M. Jaklevic
Human Genome Center and Engineering Division; Lawrence Berkeley Laboratory;
Berkeley, CA 94720
510/486-7199, Fax: -5857, Internet: wfkolbe@lbl.gov
Patricia A. Medvick, Robert M. Hollen, Tony J. Beugelsdijk, Randy S. Roberts, David M. Trimmer, Leonard A. Stovall, and Mark A. Kozubel
Los Alamos National Laboratory; Los Alamos, NM 87545
505/667-2676, Fax: /665-3911, Internet: pm@lanl.gov
Donald C. Uber, Joseph M. Jaklevic, and Edward H. Theil
Human Genome Center and Engineering Division; Lawrence Berkeley Laboratory; University of California; Berkeley, CA 94720
510/486-6378, Fax: -6816