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.

Figure 1. Figure shows the general process of probe ligation followed by FLP size-exclusion purification.

To make each probe, the variable, bridge and common strands are first combined in the presence of a ligase mix (e.g. Fast-Link or Quick Ligase); the reaction is then carried out at RT for 10 min. Afterward, the sample is passed through P-10 polymeric resin where unincorporated species are thus retained, and FLP eluted. The final purified, desalted product of pooled ODN probes is now ready for downstream application.

Figure 2. Chromatograms comparing purity and yield of final product before and after purification.

A = unincorporated common and variable strands, B = FLP, C = S1–S6 after size-exclusion purification, and D = S1–S6 after gel extraction.