Cooperative Oligonucleotides in Purification of Cycle Sequencing Products

Abstract

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.

Vascular Gene Expression in Atherosclerotic Plaque-Prone Regions Analyzed by Representational Difference Analysis

“…Colonies were PCR screened with vector-specific primers [13], and cycle sequencing was accomplished using a vector-specific primer (5)-CTA TGA CCA TGA TTA CGC CAA G-3)), and DYEnamic™ ET terminator cycle sequencing kit (Amersham Life Science, Cleveland, Ohio, USA). The cycle sequencing products were purified using solid-phase capture with cooperative probes, as previously described [14], and loaded onto a MegaBACE 1000 DNA Sequencer (Amersham Life Science). The obtained sequences were compared by BLAST [15] to the representative nucleotide sequences included in UniGene build 100 (http://www.ncbi.nlm.nih.gov/UniGene/ Mm).…”

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

“…Still, it is a robust method, and it is much cheaper then more elaborate methods based on size-exclusion chromatography and filtering. A paper by A. Blomstergren et al [16] refers to a purification method based on the use of magnetic beads. In this method, sequencing products are captured by two cooperative probes via hybridization.…”

Development of an integrated automation system with a magnetic bead‐mediated nucleic acid purification device for genetic analysis and gene manipulation

“…Biological macromolecules or cells are captured by magnetic beads, which are surface-modified or conjugated with specific ligands and then recovered by magnetic separation. The process enables a rapid change of buffers or reagents and also the concentration of nucleic acids, proteins, or cells without the use of a centrifuge (Blomstergren et al, 2000;Cox et al, 1998;Deggerdal and Larsen, 1997;Hultman et al, 1989Hultman et al, , 1991Leren et al, 1993;O'Meara et al, 1998a, b;Ståhl et al, 1999;Wahlberg et al, 1990).…”