Comparison Study Of Primer Design Strategies For Improving Specificity Of PCR Reactions

As an important molecular biology technology,real-time fluorescence PCR is widely used in biology and medicine because of its advantages of simplicity,rapidity,accuracy,no post-PCR processing,and easy automation.However,non-specific amplification in the PCR reaction is the biggest obstacle to further increase the sensitivity and specificity of the PCR.Therefore,we compared and studied the primer design strategies for PCR non-specific amplification.The first chapter described the type of non-specific amplification in the PCR reaction and the reasons for their formation,effects and solutions are described.Then,we proposed the purpose and content of this thesis.In the second chapter,we compared the results of the DPO technology and the HAND system in suppressing primer-dimers in monoplex and multiplex PCR reactions,and found that the HAND system is better than the DPO technology on suppressing primer-dimers,but HAND system also suppressed the amplification of the shorter target fragments.Then we investigated the influence of the HAND system on amplifying different fragments which own different lengths.We fund that HAND system suppressed the amplification of the target fragments smaller than 310 bp.Thence,we examined different kinds of tags and annealing temperatures.The amplific efficiency of targets can be increased without diminishing the effect of suppressing primer-dimers when we use the tags that are several bases less than the tail sequence on 5’ end used by the tailed primers and the annealing temperature that is initially low(depend on the annealing temperature of the specific sequence within the tailed primer)and then high(close but can’t be greater than or equal to the Tm of Tag sequence).At last,the Tag sequence itself may produce non-specific products,so it is best to investigate whether the Tag sequence itself will produce non-specific products first when use the HAND system.In the third chapter,the five kinds of primers that can increase the 3’-specificity of primers were compared in the first section including SuperSelective primer,DPO primer,ARMS primer,Looping out primer and DPO-A primer.We discovered that the SuperSelective primer,the Looping out primer and the mismatched ARMS primer were specific.Then the sensitivity and specificity of these three primers were compared.The sensitivity of both the SuperSelective primer and the mismatched ARMS primer was 3 copies and the selectivity was 0.01%;The sensitivity of the Looping out primer was 30 copies and the selectivity was 0.05%.In the second section,we described the feasibility of multiplex PCR detection using SuperSelective primer combined with two-dimensional labeling technique when the interrogating nucleotides close to each other and far apart from each other.