Development of automated gene diagnoses using capillary electrophoresis

我们开发了一种方法，利用非凝胶筛分毛细管电泳技术分析转化生长因子- β (tgf - β) II型受体基因富含半胱氨酸区域内的多腺嘌呤区(a) _<10>重复序列，并将其应用于结直肠癌的DNA诊断。该方法采用非对称PCR技术和毛细管电泳对(A)_<10>重复序列进行单链DNA扩增。反应混合物中dATP浓度越高，(A)_<10>重复序列的特异扩增率越高。在最佳电泳条件下，8 ~ 32个核苷酸之间可以区分出1个核苷酸的差异。在这些条件下，在30分钟内可以检测到结直肠癌中(A)_<10>重复序列的一个或两个碱基缺失，结果与DNA测序分析结果一致。根据敏感性研究，如果缺失序列出现在12.5%或更多的野生型等位基因中，我们可以检测到缺失序列。该技术的重现性令人满意，因为法内不精密度（CV） （n=10）为1.4%。这些结果表明，与传统的凝胶电泳分析相比，毛细管电泳分析小重复序列更容易处理，自动化程度更高。

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.