Development of automated gene diagnoses using capillary electrophoresis

我们开发了一种利用非凝胶筛选毛细管电泳法分析转化生长因子-βII型受体基因半胱氨酸富集区内的多聚腺嘌呤重复序列的方法，并将其应用于结直肠癌的DNA诊断。该方法包括利用不对称聚合酶链式反应技术和毛细管电泳法对(A)10&gt；重复序列进行单链DNA扩增。聚合酶链式反应混合物中的dATP浓度越高，对(A)_lt；10&gt；重复序列的特异性越强。在最佳的电泳条件下，可以在8~32个核苷酸中检测到一个核苷酸的差异。在此条件下，可在30min内检测到结直肠癌中1~2个碱基缺失，并与DNA测序结果一致。根据敏感性研究，如果在12.5%或更多的野生型等位基因中存在缺失序列，则可以检测到该缺失序列。方法重现性好，批内不精密度(n=10)为1.4%。这些结果表明，与传统的凝胶电泳法相比，毛细管电泳法对小重复序列的分析具有操作简单、自动化程度高等优点。

We developed a method to analyze a polyadenine tract, the (A)_<10> repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)_<10> repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)_<10> repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)_<10> repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyzes. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n=10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, com-pared with conventional gel electrophoretic analysis.