Resource Development 



DNA sequence data output. Genetically modified T7 DNA polymerase lacking 

 3' to 5' exonuclease activity was used in the presence of manganese ions 

 (Mn-'*) to catalyze the four separate polymerization reactions prior to sequencing 

 the vector, M13 mp18 DNA, on an Applied Biosystems Model 370A Automated 

 Sequencing System. Each polymenzation reaction was earned out with a primer 

 labeled with a different fluorophore and a different dideoxynucleoside 

 triphosphate chain terminator [Smith et al., Nature32^, 674 (1986)]. After 

 completing the polymerization reactions, the mixtures were combined and run in 

 a single lane on a denaturing polyacrylamide gel. The fluorescently labeled 

 bands separated during electrophoresis were analyzed using the Applied 

 Biosystems 370A. The red. blue, green, and black peaks correspond to ddTMP-. 

 ddCMP-, ddAtVlP-, and ddGMP-terminated fragments, respectively. Shown is a 

 part of three consecutive panels of a 450-nucleotide region of Ml 3 mpl 8 DNA; 

 the sequence of the first 20 nucleotides is TCGTACTCTAGAGGATCCCC. The 

 height of the signal decreases with the length of the fragment, because 

 statistically there are fewer terminations at each position with increasing length 

 of the DNA strand being sequenced. 



Because native T 7 DNA polymerase has an inherent 3' to 5' exonuclease 

 activity that causes premature terminations of polymerizations at pause sites, 

 T7 DNA polymerase has been modified genetically so that it no longer has 3' to 

 5' exonuclease activity. However, when the genetically modified T7 DNA 

 polymerase discriminated against the dideoxynucleoside triphosphates, uneven 

 band intensities resulted when the usual polymerization reaction component — 

 magnesium (Mg-*)— was present. Substituting Mn-* in the polymerization 

 reaction alleviated the problem of nonuniform DNA band intensity [Tabor and 

 Richardson, Proc. Natl. Acad. Sci. USASe. 4076 (1989)]. (Photograph provided 

 by S. Tabor and C. C. Richardson, Harvard Medical School.) 



