补体C3基因敲除小鼠基因型鉴定的PCR条件优化

目的对补体C3基因敲除小鼠基因型鉴定的PCR反应体系及扩增程序进行优化,检测PCR优化体系的适用性和灵敏性。方法通过改进引物设计、改变PCR反应过程中Mg2＋浓度、引物浓度、退火温度、模板DNA浓度及反应循环次数,分析比较PCR扩增效果。结果优化后的补体C3基因敲除小鼠基因型鉴定的PCR反应体系中,Mg2＋浓度在2-4mmol/L,引物浓度在0.025-0.4μmol/L,循环次数在30-45次之间均能扩增得到清晰均一的特异性条带;同时,引物退火温度在55.5℃-69.6℃范围内均适用;优化后PCR体系能够检测的最低DNA浓度为1.41ng/μl,即模板DNA在反应体系中的总量为约2.82ng.结论已经建立的优化后的PCR基因扩增体系特异性高且稳定可靠。

Optimization of PCR Conditions for Identification of Genotype of Complement C3 Knockout Mice

Objective To establish a stable PCR system by optimizing the factors that affect the sensitivity,specificity and stability of PCR,then test its applicability and sensitivity.Methods Primers were designed to identify the genotype of C3 gene knock out mice.Different PCR systems was set up by altering the factors,such as primer design and concentration,Mg2＋ concentration,annealing temperature,concentration of DNA template,and cycles.Result In the optimized PCR system,specific,sensitive and stable amplified bands were obtained when Mg2＋ concentration was between 2 to 4 mmol/L,primer concentration between 0.025 to 0.4 μmol / L,and cycles between 30 to 45 times.The range of annealing temperature was from 55.5℃ to 69.6℃.The lowest DNA concentration detected was 1.41 ng/μl,that the total amount of DNA was about 2.82 ng.Conclusion The optimized PCR reaction system has high specificity and reliability.