Department of Chemical and Biomolecular Engineering (BK21 program) and Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Electrophoresis. 2011 Jun;32(12):1456-64. doi: 10.1002/elps.201100073. Epub 2011 May 30.
We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead-based solid-phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.
我们展示了一种新型串联重复微卫星分型的概念验证，称为无 PCR 数字 VNTR（可变数串联重复）分型，它由三个步骤组成：连接反应代替 PCR 热循环、基于磁珠的固相捕获用于纯化，以及延长样品堆积微毛细管电泳 (μCE) 用于重复数的敏感数字编码。我们设计了一个 16 个碱基对的荧光标记连接探针，它与生物素化合成模板的重复单元互补，模拟人类 D1S80 VNTR 基因座，并与微卫星串联重复随机杂交。然后进行快速等温连接反应，将 DNA 模板上的相邻连接探针连接起来，然后通过链霉亲和素包被的磁珠对连接产物进行纯化。变性后，释放并回收大量大小与重复单元相当的连接产物。通过微器件上的延长样品堆积 μCE 分离，在电泳图谱中产生连接产物的荧光信号，并直接计数峰数，与 VNTR 基因座的重复数完全匹配。我们仅用 5 fmol 的 DNA 模板在 30 分钟内成功识别了微卫星串联重复数。
