Objective To establish optimal amplification conditions with the application of 0.2mL test tube in single cell separation and inspection. Methods Oral epithelium cell suspension was prepared. Five or ten cells were collected with 0.2 mL test tube. Then DNA were amplified with Identifier? Plus kit in three different conditions in which the proteinase K addition, gold enzyme concentration in PCR reaction, and PCR reaction cycles were adjusted separately. Finally the detection rate, allelic dropout rate and artificial alleles were compared among the groups. Results In these 3 different conditions: addition of proteinase K, addition of 0.4 μL gold enzyme in PCR reaction, and use of 32 cycles, the detection rate was higher and allelic dropout rate was lower than the other conditions. Conclusion In single cell separation and inspection, the usage of 0.2 mL test tube could be an effective supplement to chip-low volume amplification.%目的 优化在单细胞分离检验中应用0.2 mL试管做低体积扩增载体的最佳反应条件.方法 制备理想口腔上皮细胞悬液,用0.2 mL试管分别收集捕获到的5、10个细胞,设置蛋白酶K添加、PCR反应金牌酶用量、循环次数3组条件,用Identifiler(R) Plus试剂盒进行复合扩增,比较各组的检出率、等位基因丢失率和非特异性扩增情况.结果 用0.2mL试管做低体积扩增中,加蛋白酶K裂解、PCR反应金牌酶0.4 μL、PCR反应32个循环,这3个条件的检出率较高,等位基因丢失率较低. 结论 在单细胞分离检验中采用0.2 mL试管进行低体积扩增,可以作为芯片-低体积扩增的有效补充手段.
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