Abstracts: 



Sequencing 



Technologies 



DNA Sequencing Using Stable Isotopes 



K. B. Jacobson. H. F. Arlinghaus,* G. M. Brown. R. S. Foote. F. A. Larimer, 



R. A. Sachleben. N. Thonnard.* and R. P. Woychik 



Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077 



(615)574-1204. FTS 624-1204 



*Atom Sciences, Inc. 



Thi.s project utilizes a new DNA-sequencing approach that could increase the rate of 

 sequence determination 100-t'old or more as compared to current methods that use 

 radioisotopes. In this procedure, stable isotopes of sulfur, tin, iron, mercury, and other 

 elements can be used to label DNA it.self or oligonucleotide probes that will be used to 

 locate DNA fragments after electrophoresis. Resonance ionization spectroscopy (RIS) 

 will be used for localization and quantitation of these elements and all their stable 

 isotopes in an optimal fashion. The sensitivity of RIS, as implemented in Sputter- 

 Initiated Resonance Ionization Spectroscopy (SIRIS), should be comparable to that of 

 radioisotopes and fluorescent labels that are in current use. Furthemiore. the multiplex 

 method of DNA sequencing will be adapted for use with stable isotopes so that 40 

 labels can be used simultaneously. This adaptation will require that the maximum 

 number of stable isotopes of a given element be attached to a series of oligonucleotides 

 in a stable configuration, so that the label does not interfere with accurate hybridization. 

 Several chemical approaches are presented that should lead to properly labeled probes 

 that meet these criteria. Current state-of-the-art SIRIS will be used to demonstrate this 

 new sequencing approach, and the design of the instrument will be modified to adapt it 

 specifically to DNA sequence determination. The use of radioisotopes is eliminated 

 along with the attendant problems related to radiation exposure of personnel, the 

 prohibitive costs of radioactive waste disposal, and the need to cope with the short half- 

 lives of reagents that contain radioisotopes. A patent application is pending for this 

 technique. To carry out the goals of this project will require close collaboration of 

 physicists, chemists, and molecular biologists. 



92 



