High-Resolution DNA Mapping by Scanning Transmission 

 Electron Microscopy (STEM) 



James F. Hainfeld. Martha N. Simon, Stephen G. Will 



Biology Department, Brookhaven National Laboratory, Upton, NY 1 1973 



(516) 282-3372, FTS 666-3372 



Mapping DNA directly with STEM may complement the fast-growing technology of 

 automatic sequencers. Several tests will be made to determine the feasibility, speed, and 

 reliability of this method. There are several advantages of a direct physical microscope 

 approach to sequencing: ( 1 ) very long sequences could be done (i.e., lO'^-lO^ bp in 

 length); (2) if successful, the method could be several orders of magnitude faster than 

 chemical methods: and (3) since long pieces of DNA are used, the problems 

 encountered with repetitive sequences would be circumvented. Preliminary results have 

 been obtained using the following test system. A 622-bp sequence from pBR322 was 

 excised with restriction enzymes and purified. Next, a 128-bp T7 piece was inserted at 

 position 276 (giving a total of 720 bp). The denaturing and renaturing of equal 

 quantities of the 622-bp and 720-bp fragments resulted in 50'7f fonnation of hetero- 

 duplexes (one 622 strand paired with a 720 strand) and left the extra bases as a single- 

 stranded loop. A 26-mer oligonucleotide that was complementary to a region of the 

 single-stranded insert was synthesized. A chemical modification added a sultTiydryl at 

 the 3 -end of this oligonucleotide, and the undecagold cluster was covalently attached to 

 it. The oligonucleotide and heteroduplexes were then mixed under renaturing conditions 

 and examined using STEM. Control heteroduplexes with no gold clusters show a kink 

 at the position of the 128-bp single-stranded insert, and the total length and length to the 

 insert are consistent with the proposed model. When the gold-oligonucleotide was 

 hybridized, the gold cluster was visible as a tiny bright dot at the "V vertex of the 

 DNA. The gold cluster was about 10 A from the base it labels (3 bp), and the accuracy 

 of positioning a base from the end of DNA segments with STEM is 2 bp. A total 

 potential positional accuracy of 3-5 bp should prove useful in the physical mapping of 

 genomes. 



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