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目的改进常规的基因分型技术,以更准确、直观地鉴定芳香烃受体敲除(aryl hydrocarbon receptor knockout,AhR KO,AhR-/-)小鼠。方法剪取AhR杂合子(heterozygous,+/-)后代仔鼠脚趾,常规提取基因组脱氧核糖核酸,分别采用传统和改进的PCR方法对其基因型进行鉴定,并对结果进行比较,同时分析在试剂总量不变的情况下两种方法获得的基因分型结果的准确性和直观性。结果在反应体系调整为10μL、退火温度和时间改为65.5℃40 s、其余PCR参数(预变性温度和时间为94℃3 min;变性和延伸温度及时间分别为94℃30 s,72℃1 min,该步骤循环35次;最后一步延伸温度和时间为72℃5 min;样本保存温度为4℃)不变的情况下,采用双体系运转的改进PCR法结果比传统PCR法直观,而且总的试剂用量与传统PCR法相等。结论改进的PCR法可以更好地鉴定AhR KO小鼠表型,适合在基因分型工作中推广。
Objective To improve the conventional genotyping technique to identify more accurately and intuitively the aryl hydrocarbon receptor knockout (AhR KO, AhR - / -) mice. Methods The offspring of offspring of AhR heterozygous (+ / -) were cut off and genomic DNA was extracted routinely. The genotypes were identified by traditional and improved PCR methods respectively. The results were compared. The accuracy and intuition of the genotyping results obtained by the two methods in the case of a constant amount of total. The results were as follows: the reaction system was adjusted to 10 μL, the annealing temperature and time to 65.5 ℃ 40 s, the other PCR parameters (pre-denaturation temperature and time of 94 ℃ 3 min; denaturation and extension temperature and time were 94 ℃ 30 s, 72 ℃ 1 min, the cycle of this step 35 times; the last step of the extension temperature and time of 72 ℃ 5 min; sample preservation temperature of 4 ℃) the same circumstances, the use of improved dual PCR method PCR results more intuitive than the traditional PCR method, and the total The amount of reagents and traditional PCR method equal. Conclusion The improved PCR method can better identify the phenotype of AhR KO mice and is suitable for the promotion of genotyping.