一种新的引物二聚体形成机制  

A New Mechanism for Primer Dimer Formation

作　　者：刘姗姗[1] 岳素文 江洪 王成彬[3] 吕建新[1] 

机构地区：[1]温州医科大学检验医学院,生命科学学院,温州325000 [2]北京泰格瑞分子检验有限公司,北京102209 [3]中国人民解放军总医院临床检验科,北京100853

出　　处：《华中科技大学学报（医学版）》2014年第1期53-58,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81071426);军队“十二五”重大专项资助项目(No.AWS11Z005-4)

摘　　要：目的 通过SYBR GreenⅠ实时荧光定量PCR无模板测试技术,阐明引物二聚体形成的机制,解决PCR反应中引物二聚体形成的问题,推动SYBR GreenⅠ实时荧光定量PCR技术的应用.方法采用 SYBR GreenⅠ实时荧光定量PCR技术检测不同特点的典型引物对形成引物二聚体的Ct值,检测特异的反义核苷酸、短寡核苷酸对二聚体的影响,测试优化方案在HBV DNA定量检测中的效果.结果 SYBR GreenⅠ实时荧光定量PCR测试发现,3′端连续6～7碱基反向互补的引物对的Ct值：5.0、10.0、11.0;与另一引物中部连续6个碱基反向互补的Ct值：30.0、28.0、29.5;与另一引物5′末端连续6个碱基反向互补的引物对的Ct值：32.0、29.5、31.0.普通引物对测试的Ct值：27.5、29.0、31.0;中间部分同向相同的引物对测试的Ct值：36.5、43.0、39.0;同向相同的引物对未形成二聚体,反向相同引物对测试Ct值是30.5.低温预处理的短寡核苷酸促进二聚体形成,而在热启动PCR反应中无作用,特异的反义核酸抑制二聚体.PCR增效剂推后普通引物对的二聚体1个循环,推后部分同向相同的引物对的二聚体10个循环.中间部分相同的引物对检测低浓度质粒标准品比普通引物对检测结果更精确.结论 提出一种新的引物二聚体形成机制：引物对在3′末端碱基反向互补结合的基础上,借助剩余序列的同向互补力量拉近彼此在空间上的距离,从而进行延伸反应形成引物二聚体.中间偏3′端同向相同的引物对可以有效抑制引物二聚体,并且该作用可以被其他优化方法加强.Objective To detect the primer dimers（PDs）by using SYBR Green I real-time fluorescent quantitative PCR （SGI qPCR）without templates in order to elucidate the mechanism of the formation of PDs, raise solutions to PD formation and promote the amplification of SGI qPCR. Methods The Ct value of typical primers for the formation of PDs was detected by u- sing SGI qPCR as well as the impact of specific antisense nucleotides and short oligonucleotides on PD formation. The efficacy of the optimal proposal was evaluated in the HBV DNA quantitative measurement. Results SGI qPCR revealed that the Ct values of primers with 6 or more bases reversely complementary to each other in the 3＇end were 5.0,10.0,11.0, those reversely com- plementary to the other primer＇s middle domain were 30.0,28.0,29.5 and those corresponding to 5＇end were 32.0,29.5 and 31.0. The Ct values of ordinary primers were 27.5,29.0 and 31.0 and they were 36.5,43.0,39.0 when partial-homo-primers were used. No PDs were found between homo-primers when they were detected at 30.5 cycles using primers with opposite di- rection of base sequence. Low temperature pretreatment for short oligonucleotides contributed to the formation of PDs but played no role in hot start PCR cycles. Specific antisense nucleic acids could inhibit the formation of PDs. Moreover, the partial- homo-primers could eliminate PDs,which could be reinforced by the antisense nucleic acid or PCR enhancer. The PCR synergist made ordinary primers 1 cycle back and 10 cycles back by employing partial-homo primers. When low concentration samples were detected,partial-homo primers were more accurate than ordinary pairs. Conclusion The designed primers conforming to the general design principles can form PDs on the base of 1--2 bases reverse complementary in the 3 ＇end. They can rely on the mutual complementary power of the residual sequences, making them close to each other. Partial-homo-primers in the intermedi- ate domain can eliminate the PDs,which can be optimized by other methods.

关 键 词：SYBR GreenⅠ 引物二聚体 形成机制 

分 类 号：Q7[生物学—分子生物学]