Nucleic Acids Research, 1992, Vol. 20, No. 18 4831-4837
© 1992
METHODS |
Detection of single base differences using biotinylated nucleotides with very long linker arms
The Du Pont Merck Pharmaceutical Company Experimental Station, P0 Box 80328, Wilmington, DE 19880-0328, USA
Received June 8, 1992. Revised August 12, 1992. Accepted August 12, 1992.
A simple primer extension method for detecting nucleotide diflerences is based on the substitution of mobility-shifting analogs for natural nucleotides (1). This technique can detect any single-base diflerence that might occur including previously unknown mutations or polymorphisms. Two technical limitations of the original procedure have now been addressed. First, switching to Thermococcus littoralis DNA polymerase has eliminated variability believed to be due to the addition of an extra, non-templated base to the 3' end of DNA by Taq DNA polymerase. Second, with the analogs used in the original study, the mobility shift induced by a single base change can usually be resolved only in DNA segments 200 nt or smaller. This size limitation has been overcome by synthesizing biotinylated nucleotides with extraordinarily long linker arms (36 atom backbone). Using these new analogs and conventional sequencing gels (0.4 mm thick), mutations in the human ß-hexosaminidase 
and CYP2D6 genes have been detected in DNA segments up to 300 nt in length. By using very thin (0.15 mm) gels, single-base polymorphisms in the human APOE gene have been detected In 500-nt segments.