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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 single cell-level method for detecting single nucleotide polymorphisms using a microfluidic chip as a detection platform. Methods: Single lymphocytes were isolated from venous blood of healthy adults. Single cell template was prepared by different cell lysis methods. The target fragment of CYP2B6 gene and restriction enzyme digestion products were amplified by nested PCR. Using conventional agarose gel electrophoresis and microfluidic chip method for testing. Results: The success rates of single-cell DNA template amplification by enzymatic cleavage method, alkaline cleavage method and freeze-thaw method were 96%, 85% and 72%, respectively. The genotyping results of single-cell PCR micro- Sugar gel electrophoresis method is exactly the same. Conclusion: The single-cell PCR-based microfluidic chip method can detect CYP2B6 genotypes of single lymphocyte specifically and accurately, and the sample consumption is low, the operation is simple and the results are reliable.