Engelstein M, Aldredge T J, Madan D, Smith J H, Mao J I, Smith D R, Rice P W
Genome Therapeutics Corp., Waltham, Massachusetts, USA.
Microb Comp Genomics. 1998;3(4):237-41. doi: 10.1089/omi.1.1998.3.237.
We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.
我们开发了一种适用于96孔板的DNA模板分离方法，该方法易于自动化操作。模板分离方案涉及简单的碱性裂解化学过程以及在硅胶固相上的可逆捕获。细胞裂解后，无需离心，因为裂解物纯化、DNA结合、洗涤和释放均在96孔滤板中进行。使用硅胶纯化方法制备的大量模板已进行测序和分析。我们将该方法得到的序列质量与几种商业质粒制备方案所产生的序列质量进行了比较。我们发现硅胶珠制备物的序列质量与其他方法制备的相当，在某些情况下甚至更好。该方法比我们使用过的其他方案具有许多优势。首先，硅胶纯化使我们的整体实验室通量提高了一倍多，同时将模板分离材料成本降低了至少五倍。其次，由于我们在方案中省去了所有离心步骤，自动化操作变得更加简单。该方案还经过调整，可用于纯化PCR产物，以便在后续测序反应中用作模板。
