A solid-phase method for the purification of DNA sequencing reactions
A solid phase method for the purification of 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 at 90[degrees]C, and may then be directly loaded into a polyarcylamide gel for sequence analysis. This method was used to investigate the effect of various contaminants such as template DNA, salts and glycerol upon DNA sequence data. It is found that signal intensity and base calling accuracy are all increased by the elimination of template DNA, glycerol, and salts. The potential for automation of this solid phase method for large-scale DNA sequencing is discussed.
- Research Organization:
- Wisconsin Univ., Madison, WI (United States)
- OSTI ID:
- 7105254
- Country of Publication:
- United States
- Language:
- English
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