Stanford Genome Technology Center, Palo Alto, California, United States of America.
PLoS One. 2012;7(4):e34373. doi: 10.1371/journal.pone.0034373. Epub 2012 Apr 6.
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.
在这里，我们介绍了一种快速、经济有效的方法，只需不到 15 分钟的时间即可生成分子 DNA 探针，而无需昂贵且耗时的凝胶提取步骤。例如，我们使用 Fast-Link DNA 连接酶将六个可变链（50bp）与一个共同的链序列（51bp）在单个池中共轭，以产生 101bp 靶标（10 分钟）。然后，通过将粗反应液通过排阻层析柱（<5 分钟）过滤掉未结合的物质。然后，我们使用 HPLC 分析比较了粗样品和纯化样品的全长产物产量；结果清楚地表明，我们的方法产量是粗样品的四分之三（比凝胶提取高 50%）。虽然我们通过过滤过程大大减少了未连接产物的量，但通过进一步优化，可以实现更高的纯度和产量，每个反应的链数增加（>12）。此外，对于大规模分析，我们设想可以使用机器人（如 Biomek FX）对此方法进行完全自动化，在此情况下，可在数分钟内将数千个样品在 96、384 或 1536 孔板中进行池化、连接和纯化。
