Abstracts: 



Mapping 



Instrumentation 



Human Genome Center, Lawrence Berkeley Laboratory 



Charles R. Cantor, C. Bustamante, J. Gingrich, A. Hassenfeld, J. Jaklevic, 

 W. Johnston, J. Katz, W. F. Kolbe, S. Levene, S. Lewis, M. Maestre, and E. Theil 

 Human Genome Center, Lawrence Berkeley Laboratory. Berkeley, CA 94720 

 (413)486-6800, (FTS) 451-6800 



Researchers at the Human Genome Center at Lawrence Berkeley Laboratory (LBL) are 

 developing innovative techniques in instrumentation and automation to accommodate 

 the size and complexity of the experimental procedures used in physical mapping 

 methods. In addition to improving existing laboratory methods, emphasis will be placed 

 on developing advanced techniques for separating large DNA fragments. Technology 

 for the flow separation of chromosomes will be developed. Modem nuclear radiation 

 detectors or optical and ultraviolet imaging systems will be used to explore methods for 

 direct imaging of electrophoresis gels. The use of differential-polarization imaging to 

 achieve enhanced sensitivity for direct viewing of DNA will be investigated. 



Brief abstracts of the individual projects are listed below. 



Optimization of Pulsed-Field Gel Electrophoresis (A. Hassenfeld, J. Jaklevic. J. 

 Katz, W. F. Kolbe, and S. Levene) — Engineers at LBL have constructed a test bed for 

 all of the various configurations of PEG electrophoresis. This test bed provides 

 precision control and recording of the conditions within the gel in real time during the 

 run. Optimizing the speed and precision of DNA isolation will, in turn, shorten crucial 

 steps in the mapping process while retaining the high resolution of the current PEG 

 techniques. Among the variables currently being explored are short, intense secondary 

 electric field pulses and combined electric and magnetic fields. 



Micromanipulation of DNA (M. Maestre and C. Bustamante) — Some of the 

 infomiation from the PEG studies is being applied to the development of a system for 

 handling and manipulating single DNA molecules. The goal is to develop techniques for 

 isolating specific, single DNA molecules for other procedures such as PCR. cutting by 

 enzymatic or physical means, or direct visualization. The rationale for this technique is 

 to construct a network of electrodes that are about the same size as large DNA 

 molecules (electrodes of 10-20 mm separated by 20-50 mm). A key concept is the use 

 of inhomogeneous fields. If a DNA molecule is to be manipulated in a way that places 

 different sections of the molecule in specific positions in the electrode net, it is essential 

 that these parts experience different forces and different electrostatic fields. The motion 

 of single DNA molecules has been made visible with the use of the intensified 

 epifluorescence microscope after labeling with fluorophores (acridine orange). Direct 

 manipulation of the molecule is then performed through the computer-assisted control 

 of the local electric fields by the microelectrodes. The microelectrodes have been 

 constructed and tested, and DNA molecules can be moved in predictable directions. T4 

 phage DNA was used in the preliminary testing. The DNA molecules stretched to a 

 length of 49 mm; this length is very close to the length of 52 mm reported for the T4 

 phase DNA. 



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