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Nucleic Acids Research, 2002, Vol. 30, No. 13 2877-2885
© 2002 Oxford University Press

Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to ‘direct-load’ DNA sequencing

Patrick J. Finn, Lei Sun, Satyam Nampalli, Haiguang Xiao, John R. Nelson, J. Anthony Mamone, Greg Grossmann, Parke K. Flick, Carl W. Fuller and Shiv Kumar*

Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo SequenaseTM II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.

* To whom correspondence should be addressed. Tel: +1 732 980 2878; Fax: +1 732 457 8353; Email: shiv.kumar{at}am.amershambiosciences.com


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P. J. Finn, M. G. Bull, H. Xiao, P. D. Phillips, J. R. Nelson, G. Grossmann, S. Nampalli, B. F. McArdle, J. A. Mamone, P. K. Flick, et al.
Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing
Nucleic Acids Res., August 15, 2003; 31(16): 4769 - 4778.
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