ACTB基因引物的设计及其在医学本科分子生物学实验教学中的应用

目的针对ACTB基因设计引物,并在医学本科分子生物实验教学中通过聚合酶链式反应(PCR)让学生认识真核基因内含子和外显子结构的特点。方法通过在线软件Primer-BLAST设计针对ACTB基因的引物,通过改变退火温度,优化PCR体系,同时,结合实验教学结果评价引物的特异性。结果针对ACTB基因外显子3和外显子4设计了两对引物,引物1和引物2。引物1在退火温度68℃时,表现出较高的特异性和PCR产量。以基因组DNA(gDNA)和cDNA为模板,PCR产物大小相差441bp。以此条件,引物1在实验教学中也表现出较好的效果,目的基因扩增成功率达到90%以上。结论引物1在退火温度68℃时,利用PCR技术结合琼脂糖凝胶电泳结果,学生们可以形象的理解真核生物基因的结构特点,值得在医学本科分子生物学实验教学中推广。

Objective To design specific primer for ACTB and apply it in the molecular biology experiment teaching for medical undergraduate to help them understand the structure characteristics of eukaryotic genes with intron and exon through PCR.Methods ACTB primers were designed using online tool Primer-BLAST.PCR system was optimized by different annealing temperatures.Primer specificity was evaluated based on experiment teaching results.Results Two ACTB primers(primer 1 and primer 2)were designed spanning exon 3 and exon 4.Primer 1 showed high specificity and PCR yield at annealing temperature of 68℃.There was 441 bp difference of PCR product between genomic DNA(gDNA)and cDNA as template.Under this condition,we also obtained a satisfied experiment teaching result for primer 1,with more than 90%successful amplification rate of target gene.Conclusion Students can easily understand the structural characteristics of eukaryotic genes by PCR using primer 1 at annealing temperature of 68℃,which is worth popularizing in the molecular biology experiment course for medical undergraduates.