Physical Mapping 



Automated cosmid fingerprinting and contig assembly. Chromosome-19- 

 speclfic cosmids are digested with restriction enzymes, and thie fragments are 

 labeled with fluorochromes. The Beckman Instruments, Inc., Biomek «' robotic 

 system processes sets of 48 cosmids per experiment. Throughput is increased, 

 because of the capability to load the restriction fragments from three cosmids (each 

 labeled with a different fluorochrome) plus size standards (a fourth fluorochrome) in 

 each lane of a denaturing polyacrylamide gel. The labeled restriction fragments are 

 detected, and the fluorescence is digitized as the fragments migrate past a laser 

 beam in an Applied Biosystems 370 automated DNA sequencer. Fragment data 

 acquisition may be monitored during electrophoresis to ensure that operation is 

 proceeding normally. 



In the large photograph, the fragment peaks from each of three cosmids are 

 labeled in blue, green, and yellow. The size standard is in red. Several lanes of the 

 gel are depicted on the top of this figure, and a historical view of the data from one 

 lane is shown in the lower plot. The restriction fragment mobility data for each 

 cosmid are analyzed by a suite of software programs developed at Lawrence 

 Livermore National Laboratory. Fluorescence signals are processed to remove 

 noise, to identify peaks (representing restriction fragments), and to calculate 

 fragment lengths by comparison to the size standards. 



The inset photograph demonstrates fragment size comparisons for all pairs of 

 cosmids to determine whether they share a significant number of fragments of the 

 same size (to within 1-1 .5 bases). A single statistic is calculated that estimates the 

 strength of the overlap. A best-overlap solution is determined and presented 

 graphically for inspection, manipulation, and detailed query of the underlying 

 database. The cosmids, represented by the warmer-color (red and yellow) bars, 

 are those that exhibit the most overlap. (Photographs provided by Anthony 

 Carrano, Lawrence Livermore National Laboratory.) 



30 



