Sequencing of Linear Molecules 



Joseph M. Jaklevic and W. F. Kolbe 



Instrumentation Division. Lawrence Beri<eiey Laboratory. Berkeley. CA 94720 



(4LS) 486-.'^647. FTS 431-5647 



Innovative methods for determining the linear sequence of nucleic acid bases along 

 mapped fragments of human chromosomal DNA are required for the efficient 

 implementation of human genome sequencing. This project involves the investigation of 

 physical analytical methods for determining the base sequence of DNA. The initial 

 approach focuses on the development of techniques for manipulating individual DNA 

 molecules or ordered arrays of identical molecules in a manner that will allow 

 sequencing of the individual bases using direct spectroscopic methods. An intermediate 

 step will be an attempt to combine the manipulation of ordered arrays with solid-phase 

 restnction enzyme chemistry to achieve an advanced method for physical mapping of 

 intennediate-sized fragments. Since the spatial resolution and analytical sensitivity 

 required for DNA sequencing are significantly beyond existing capabilities, the 

 feasibility of combining several innovative technologies will be explored. Initially, we 

 will investigate low-temperature solid matrices as media for both physical manipulation 

 and chemical isolation of individual molecules. Subsequent alignment of the DNA 

 strands using electromagnetic fields will be investigated using direct imaging methods 

 such as scanning tunneling microscopy and fluorescence microscopy of labeled 

 molecules. Methods for attaching cloned DNA fragments to appropriate substrates will 

 also be incorporated into the matrix studies. Spectroscopic methods applicable to the 

 detection of the DNA molecules at the required level of sensitivity will also be 

 explored. 



93 



