反向重复序列DNA的PCR扩增特性研究

PCR Amplification Characteristics of DNA with Inverted Repeat Sequence

旨在研究反向重复序列形成的发卡结构对模板扩增的影响。研究DNA发卡结构中环部大小、茎部长度、引物相对于茎部位置等对PCR扩增效率的影响,探讨如何通过引物设计及改变退火温度等条件来实现发卡结构DNA的有效扩增的方法。结果显示,如果引物的位置同发卡结构茎部5'序列相同或部分相同,则由于发卡结构的形成阻碍引物与模板结合而对PCR产生抑制作用,并且抑制作用随着茎部长度的增加而增强;当环部的长度达到50 nt以上时,抑制作用明显减弱。较高的退火温度和引物浓度都有助于引物同分子内形成发卡结构的竞争,使PCR扩增更容易进行。

This work is to explore that effect of hairpin structure formed by inverted repeats on template amplification. We investigatedthe factors affecting PCR amplification efficiency of DNA with hairpin structure,including ring size,stem length and the stem position as to theprimer＇s hybridization,and discussed how to achieve efficient amplification of DNA with hairpin structure by designing primers and changingthe annealing temperature. The results showed that if the position of the primers completely or partly complemented to the 5＇-sequence of thehairpin stem,the formation of hairpin structure hindered the binding of primer and template,therefore the inhibition to PCR amplificationoccurred;moreover,the inhibition increased with the stem length increasing. The inhibition decreased significantly when,the loop portionwas longer than 50 nt. Higher annealing temperature and primer concentration were favorable for primers to compete with the formation of hairpinstructure within molecule,therefore leading PCR amplification to be more efficient.