56. Verifying Sequence By Atomic Force Microscopy 

David P. Allison and Peter R. Hoyt 
Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123 
allisondp@ornl.gov 

Atomic force microscopy (AFM) technology can be developed, as a sequencing alternative, to verify homologies between DNA species, accurately, inexpensively, and with high-throughput. 

By forming heteroduplexes between sequenced and test molecules, deletions, substitutions, and perhaps even point mutations, can be imaged and precisely located by AFM.  

Using AFM imaging we have identified deletions of 22 to 450 bp in heteroduplexes of linearized mutant and wildtype pSV-ß-Galactosidase plasmid (6821 bp). Additionally, utilizing an AFM technique we developed for mapping cosmids, which employs imaging a cleavage deficient mutant EcoRI endonuclease site-specifically bound to active sites, we have simultaneously located deletions relative to the EcoRI sites on the pSV-ß-Galactosidase heteroduplexes.  

We have imaged the specific binding of mismatch repair enzymes to heteroduplexes between wildtype and plasmids with point mutations. Conditions to maximize binding efficiency and define specificity for all combinations of mismatches are being evaluated on plasmid constructs. 

When developed this technology could provide high-throughput sequence verification of heteroduplexes generated from long range PCR or clone libraries. Furthermore these procedures can be accomplished by technicians, use readily available relatively inexpensive instrumentation, and should be fully transferable to most laboratories.