微流控芯片法检测单细胞水平SNP:以CYP2B6为例

Detection of SNP from a single cell by microfluidic chip with CYP2B6 as example CYP2B6

目的:以微流控芯片为检测平台,建立单细胞水平检测单核苷酸多态性的实验方法。方法:从健康成人静脉血中分离出单个淋巴细胞,采用不同的细胞裂解方法制备单细胞模板,应用巢式PCR方法扩增CYP2B6基因目标片段,限制性内切酶酶切产物,酶切后分别用常规的琼脂糖凝胶电泳技术和微流控芯片法进行检测。结果:酶裂法、碱裂法和冻融法制备单细胞DNA模板的扩增成功率分别为96%,85%和72%,单细胞PCR微流控芯片法基因分型结果与普通PCR琼脂糖凝胶电泳方法完全一致。结论:单细胞PCR微流控芯片法能够特异、准确地检测单个淋巴细胞的CYP2B6基因型,且样品消耗量低,操作简便,结果可靠。

Objective：To establish a method for single nucleotide polymorphism detection in a single cell by microfluidic chip and analyze the factors influencing the amplification results.Methods： The single lymph cell was isolated from whole blood using a glass capillary.Single cell DNA templates were prepared with different methods.CYP2B6 genotype were determined by a nested polymerase chain reaction-restriction fragment length polymorphisms method（NPCR-RFLP） from a single cell.Then microfluidic chip technology and agarose gel electrophoresis were used to detect SNP,respectively.Results： Enzyme lysis method is the most efficient procedure for preparing the single cell DNA template,with a success rate（SR） of 96%.The nested polymerase chain reaction technique is efficient for single cell genotyping.The chip based method is found very sensitive,requiring much less sample and only quarter of the time compared to agarose gel method.Conclusion： The NPCR-microfludic chip technology is significantly more rapid,sensitive than agarose gel electrophoresis method for detection of SNP.