Cooperative oligonucleotides in purification of cycle sequencing products.
Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.
Blomstergren
A
A
Royal Institute of Technology (KTH), Stockholm, Sweden.
O'Meara
D
D
Lukacs
M
M
Uhlén
M
M
Lundeberg
J
J
D001483:Base Sequence; D004396:Coloring Agents; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004274:DNA, Recombinant / Q000302:isolation & purification* / Q000378:metabolism; D017743:Equipment Reuse; D018189:Immunomagnetic Separation; D008863:Microspheres; D008969:Molecular Sequence Data; D009176:Mycoplasma mycoides / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic
D004396:Coloring Agents; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides