title: A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.
abstract: Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, United States of America. m.a.jensen@stanford.edu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jensen
authors.forename: Lauren
authors.initials: L
authors.lastname: Jauregui
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003362:Cost-Benefit Analysis; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000096:biosynthesis / Q000191:economics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000191:economics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis / Q000191:economics / Q000302:isolation & purification*
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases; D000072481:DNA Ligase ATP