Abstract
A solid-phase method for the purification of the single-stranded DNA molecules produced in enzymatic sequencing reactions has been developed. A primer oligonucleotide is synthesized containing a biotin moiety at an internal position. This primer is utilized in enzymatic extension reactions, and the resulting products are bound to streptavidin-coated magnetic beads. Contaminating species such as protein, salts, template DNA, and unincorporated or degraded deoxy and dideoxy nucleotide triphosphates may be removed by washing the beads after immobilizing them in the sample tube with a fixed magnet. The resulting pure single-stranded DNA fragments are removed from the solid support by heating in 10 mM EDTA, 95% formamide, loading dye at 90 degrees C, and may then be directly loaded onto a polyacrylamide gel for sequence analysis. This method was used to investigate the effect of various contaminants upon DNA sequence data.
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