Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture.
A solid-phase triple-helix-mediated affinity capture method is described for the purification of single-stranded M13 DNA for use as template in fluorescence-based DNA sequencing reactions. In this method, a biotinylated polypyrimidine oligonucleotide "loop" bound to streptavidin-coated magnetic beads is used to selectively capture single-stranded M13 DNA from high-titer phage supernatant through the formation of a cooperative triple helix (CTH) complex between the oligonucleotide and a polypurine site previously cloned into the M13 vector. Capture is accomplished at acidic pH to encourage triple-helix formation, while elution is performed at alkaline pH with heating to destroy the CTH complex. The beads can be reused up to three times without probe replenishment. Yields of M13 ssDNA in excess of 1 microgram per milliliter of culture are obtained, sufficient for use as template in fluorescence-based DNA sequencing reactions.
Johnson
A F
AF
Department of Chemistry, University of Wisconsin, Madison 53706-1396, USA.
Wang
R
R
Ji
H
H
Chen
D
D
Guilfoyle
R A
RA
Smith
L M
LM
D017104:Bacteriophage M13; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification*; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D005822:Genetic Vectors; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D001710:Biotin