Beattie K L, Logsdon N J, Anderson R S, Espinosa-Lara J M, Maldonado-Rodriguez R, Frost J D
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Biotechnol Appl Biochem. 1988 Dec;10(6):510-21.
Gene synthesis is a potentially powerful tool in molecular biology that has not yet reached widespread use because of the relatively high cost and labor-intensive nature of the process. This paper reviews some recent technological developments and current research activities of this laboratory which promise to greatly reduce the cost of gene synthesis and to increase the speed and efficiency of the process. We recently developed an improved device for "segmented" synthesis of oligonucleotides, which utilizes porous Teflon wafers containing derivatized controlled pore glass supports to simultaneously synthesize up to 100 different DNA sequences. The stepwise coupling efficiency with the "wafer synthesis device" is as high as that attained with current automated "gene machines" producing 1-4 oligonucleotides at a time, whereas the reagent usage is only 20-50% that of the current DNA synthesizers. At present, we are optimizing the conditions for rapid, efficient assembly of genes on a solid-phase support, wherein ordered, stepwise annealing/washing is performed to segmentally elongate a "starting" oligonucleotide attached to a solid-phase support. We expect that the wafer synthesis device (operated at reduced scale of synthesis), together with solid-phase gene assembly, will permit the synthesis and assembly of an average size gene (1 kb) in one week at a cost of less than $1000. These developments should make gene synthesis a routine and powerful tool in molecular biology.
基因合成是分子生物学中一种具有潜在强大功能的工具，但由于该过程成本相对较高且劳动强度大，尚未得到广泛应用。本文综述了本实验室最近的一些技术发展和当前的研究活动，这些有望大幅降低基因合成成本，并提高该过程的速度和效率。我们最近开发了一种用于寡核苷酸“分段”合成的改进装置，它利用含有衍生化可控孔径玻璃载体的多孔聚四氟乙烯晶片，可同时合成多达100种不同的DNA序列。使用“晶片合成装置”的逐步偶联效率与目前一次生产1 - 4个寡核苷酸的自动化“基因机器”所达到的效率一样高，而试剂用量仅为目前DNA合成仪的20 - 50%。目前，我们正在优化在固相载体上快速、高效组装基因的条件，其中通过有序、逐步的退火/洗涤来分段延长连接在固相载体上的“起始”寡核苷酸。我们预计，晶片合成装置（以较小规模合成运行）与固相基因组装相结合，将能够在一周内以低于1000美元的成本合成并组装一个平均大小的基因（1 kb）。这些进展应会使基因合成成为分子生物学中一种常规且强大的工具。
