Stern Rowena F, Roberts Roland G, Mann Kathy, Yau Shu C, Berg Jonathan, Ogilvie Caroline Mackie
Department of Medical and Molecular Genetics, Guy's, King's and St. Thomas' School of Medicine, London, UK.
Biotechniques. 2004 Sep;37(3):399-405. doi: 10.2144/04373ST04.
The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.
多重连接依赖探针扩增技术（MLPA）的最新进展为在单个反应中对多个位点进行剂量筛选提供了一种高效且可靠的检测方法。然而，该方法的一个缺点是通过在单链噬菌体载体中克隆来生成探针组的过程耗时。我们开发了一种合成探针组，用于筛选3号染色体q臂上跨度为18.5 Mb区域的缺失情况。在一项初步研究中，我们在4个对照样本以及2名先前被发现3q26 - q28区域存在杂合缺失的患者身上测试了15种合成探针。这些合成探针在所有先前已知的缺失位点均检测到了缺失。此外，使用合成探针时，样本内结果的变异性与市售M13衍生探针所报道的相似。我们的结果表明，这种MLPA新方法为通过克隆进行探针开发的难题提供了一个通用解决方案；此类合成产生的探针可用于在单个反应中筛选大量位点。我们得出结论，在MLPA中使用合成探针是一种快速、稳健且高效的替代方法，可用于多个基因组位点的研究（以及潜在的诊断）性缺失和重复筛选。
