循环次数与体系对DNA检测灵敏度的影响

目的探讨通过增加PCR循环次数和缩小扩增体系改善DNA检测灵敏度的可行性。方法将10例无血缘关系健康志愿者精液DNA样本分别定量为50、40、30、25、20、15、10 pg/μL,分别用10、5、3μL的体系和28、30、32、34、36次循环进行扩增,用3130遗传分析仪检测15个常染色体STR基因座。结果 28次循环下,3μL体系可对40pg/μL及以上样本正确分型。20pg/μL及以上样本在10、5、3μL体系中,34次循环均可正确分型,增加到36次循环,出现非特异性谱带,无法正确分型。结论增加循环次数和缩小扩增体系在一定程度上可以提高DNA检测的灵敏度。

Influences of Cycle Index and Volume on Sensitivity of DNA Detection

Objective To explore the feasibility of improving the sensitivity of DNA detection by increas- ing the PCR cycle index and decreasing the volume of amplifying system. Methods The DNA of semen were collected from 10 healthy irrelevant volunteers, and were quantified to 50, 40, 30, 25, 20, 15, 10 pg/μL, separately. All samples were then amplified in 10, 5, 3 μL volume and at 28, 30, 32, 34, 36 cycles, re- spectively. 3130 genetic analyzer was used to detect 15 autosomal STR loci. Results Under the situation of 28 cycles and 3 μL volume, samples which achieved 〉40pg/μL could be correctly typed. Under the situation of 10, 5, 3 μL volume, samples which achieved 〉20 pg/μL could be correctly typed at 34 cy- cles. When increasing the index to 36 cycles, they could not be correctly typed because of the non-spe- cific band. Conclusion DNA detecting sensitivity can be improved to a certain extent by increasing the cycle index and decreasing the volume of amplifying system.