单链环状DNA添加剂抑制基因表达序列分析Ditag聚合酶链反应引物多聚体生成

Using Single Strand Circular DNA Additive to Inhibit Primer Polymers Generated during Ditag PCR of Serial Analysis of Gene Expression

[目的]研究利用单链环状DNA抑制聚合酶链反应（PCR）副产物的作用效果。[方法]设计并合成单链环状DNA,并以具同样序列发卡状结构的单链DNA为对照组,在基因表达序列分析（SAGE）Ditag PCR反应中加入上述不同浓度的DNA,利用电泳技术检测不同结构（环状/单链）以及不同浓度单链DNA对PCR多聚体附产物的抑制作用。[结果]发卡状结构的单链DNA无法抑制PCR引物多聚体副产物的产生;而在70～150 nmol/L终浓度范围内,单链环状DNA可以抑制SAGE Ditag PCR反应中引物多聚体的生成,并且不影响SAGE不同tag间的表达丰度差异。[结论]单链环状DNA可以作为PCR反应中引物多聚体生成的有效抑制剂。

[Objective]To investigate the hypothesis that using single strand DNA as additive to prevent mispriming product of PCR. [Method] We designed and synthesized two kinds of single strand DNA additives; one was circular and another hairpin-like. Different concentrations of each kind of DNA additives were added into Ditag PCR systems of Long Serial Analysis of Gene Expression. Final products were used for gel electrophoresis to screen DNA additives structure-or concentration-dependent inhibition of PCR primer polymer. [Results]Single strand circular but hairpin-like DNA additives was able to inhibit PCR primer polymer; the optimal final concentration was between 70 and 150 nmol / L,and single strand circular DNA additives will not affect differential expression of SAGE tags. [Conclusion]Single strand circular DNA can be used as effective inhibitor to prevent primer polymers in PCR amplification.