Direct oligonucleotide synthesis onto super-paramagnetic beads.
Super-paramagnetic beads (SPMB)s used for a variety of molecular diagnostic assays are prepared by attaching pre-synthesized oligonucleotides to the surface via a cumbersome and low efficient method of carbodiimide-mediated amide bond formation. To mainstream the process, we describe a novel procedure of direct oligonucleotide synthesis onto the surface of SPMBs (e.g. MyOne Dynabeads). With the many challenges surrounding containment of paramagnetic beads (≤1 μm) during automated oligonucleotide synthesis, we show that by applying a magnetic force directly to the SPMBs we prevent their loss caused by high-pressure drain steps during synthesis. To date we have synthesized 40 mers using a Spacer 9 phosphoramidite (triethylene glycol) coupled to the surface of hydroxylated SPMBs. HPLC analysis shows successful product generation with an average yield of 200 pmol per sample. Furthermore, because of the versatility of this powerful research tool, we envision its use in any laboratory working with conventional synthesis automation, as employed for single columns and for multi-well titer plates. In addition to direct synthesis of oligodeoxynucleotides (DNA) onto SPMBs, this platform also has the potential for RNA and peptide nucleic acid synthesis.
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA. Electronic address: m.a.jensen@stanford.edu.
Michael A
MA
Jensen
Michael S
MS
Akhras
Marilyn
M
Fukushima
Nader
N
Pourmand
Ron W
RW
Davis
D001331:Automation / Q000295:instrumentation; D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000138:chemical synthesis; D007202:Indicators and Reagents; D060526:Magnetic Fields; D009841:Oligonucleotides / Q000138:chemical synthesis*
D007202:Indicators and Reagents; D009841:Oligonucleotides; D004247:DNA
DNA synthesis; Nucleic acid; Super-paramagnetic beads; Synthesis automation