Research Facility 

 Narratives: LLNL 



To construct a set of cosmid contigs (overlapping clones) for chromosome 19, LLNL 

 has developed a semiautomated fluorescence-based strategy for fingerprinting each 

 clone. For this procedure, researchers use a robotic system to attach fluorophores to the 

 ends of restriction fragments from each cosmid clone. Fragment lengths are determined 

 using a commercially available laser scanning device to acquire data in real time from a 

 polyacrylamide gel. The data collection is multiplexed in the sense that up to four 

 different fluorophores (i.e.. four clones) can be run in each gel lane. In this 

 conflguration, 48 cosmids can be analyzed per gel run. 



Livemiore has developed software that will process the acquired fingerprint signals, 

 convert the information to a fragment length for each cosmid, use the fragment length 

 data to compute a statistical measure of overlap between cosmids. and allow users to 

 display data graphically and browse among the cosmid contigs. The flngerprinting 

 procedure, from fluorochrome labeling through data processing and contig 

 visualization, is now automated. 



About three thousand cosmids have been processed to date. Researchers at LLNL have 

 established over 330 cosmid contigs for chromosome 19 — estimated to span 30% of the 

 chromo.some or about 20 Mbp. In addition, they established 6 cosmid contigs that span 

 approximately 600 kb of chromosome 14. Several of the chromosome- 19 contigs 

 represent known gene loci; the others are located throughout the chromosome. A 

 number of these contigs have been validated by restriction fragment digests or by in situ 

 hybridization to metaphase chromosomes. 



In a study parallel to contig map construction, Livermore is developing a large-fragment 

 restriction map of chromosome 19: this approach will eventually All the gaps between 

 contigs. LLNL researchers have discovered that two of the DNA repair genes on this 

 chromosome lie within 260 kb of each other. In addition, they have devised a technique 

 to isolate region-specific probes based upon polymerase chain reaction (PCR) 

 amplification of DNA located between human A/w-repetitive sequences. These probes 

 are being used to identify those cosmids (from Livermore "s chromosome- 19 library) 

 that span a specific region of the chromosome. 



In collaboration. Lawrence Berkeley Laboratory and LLNL have used scanning 

 tunneling microscopy to produce the highest resolution image of DNA to date. Further 

 work at Livermore is focusing on the application of this technique to DNA sequencing. 

 Advances have been made in DNA deposition techniques, construction of a new 

 computer-controlled scanning tunneling microscope, refinement of the electronics to 

 minimize noise, and development of new computer programs to improve visualization 

 and analysis of imaged DNA. 



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