Solid phase purification in automated DNA sequencing.
A solid phase purification method has been utilized to investigate the effect of template DNA and other contaminants present in enzymatic DNA sequencing reactions upon data quality in automated DNA sequence analysis. The primer oligonucleotide is prepared with an internal biotin group and one of four fluorescent tags at the 5' terminus. The fluorescently labeled DNA fragments produced in the sequencing reactions are separated from other reaction components, including template DNA, using superparamagnetic streptavidin beads. Template DNA and other reaction components may then be added back to the purified material to assess their effect upon sequence data. It is found that band intensity and base calling accuracy are decreased by the presence of the template DNA and other reaction components.
Tong
X
X
Department of Chemistry, University of Wisconsin, Madison 53706.
Smith
L M
LM
D000818:Animals; D001483:Base Sequence; D001710:Biotin / Q000138:chemical synthesis; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000138:chemical synthesis / Q000235:genetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D001710:Biotin; D004247:DNA