Department of Obstetrics and Gynecology, Daping Hospital, Third Military Medical University, Daping, Yuzhong District, Chongqing, People's Republic of China.
Cell Biochem Biophys. 2012 Jan;62(1):161-7. doi: 10.1007/s12013-011-9277-2.
We tested applicability of a new genotyping technique to detect a low abundance CD17 (A → T) mutation of β-globin gene. The technique utilized a combined gap ligase chain reaction (Gap-LCR) and quantitative PCR (qPCR) methods. One pair of Gap-LCR primers was modified by adding specific sequences to the 5' end of the upstream and the 3' end of the downstream primer which served as a combining sequence for qPCR. First, specific mutation is detected using Gap-LCR; then, ligation products are detected by qPCR. Our results show that the amount of LCR products is directly proportional to the amount of template DNA. We further demonstrate that this technique detects a low abundance mutant DNA with a mutant/normal allele ratio as low as 1:10000. This technique was applied to detect a paternally inherited CD17 mutation from 53 maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. In conclusion, by combining Gap-LCR and qPCR technology we successfully established a highly sensitive technique to detect low abundance point mutations. This technique can be applied to detect fetal DNA point mutation in maternal plasma.
我们测试了一种新的基因分型技术在检测低丰度β-珠蛋白基因 CD17(A→T)突变中的适用性。该技术结合了缺口连接酶链反应（Gap-LCR）和定量 PCR（qPCR）方法。一对 Gap-LCR 引物通过在上下游引物的 5'端和 3'端添加特定序列进行了修饰，这些特定序列作为 qPCR 的结合序列。首先，使用 Gap-LCR 检测特定的突变；然后，通过 qPCR 检测连接产物。我们的结果表明，LCR 产物的量与模板 DNA 的量成正比。我们进一步证明，该技术可以检测到低丰度突变 DNA，突变/正常等位基因比例低至 1:10000。该技术应用于检测 53 份母体外周血浆样本中的父系遗传 CD17 突变。结果与羊水细胞 DNA 的 PCR/反向斑点印迹法一致。总之，通过结合 Gap-LCR 和 qPCR 技术，我们成功建立了一种高度敏感的检测低丰度点突变的技术。该技术可应用于检测母体外周血浆中的胎儿 DNA 点突变。
