引物设计重点因素及设计技巧

想把引物合成的比较好,除了前引物和后引物的Tm不能相差太大,我们还要重点考虑以下因素:

一、GC含量

引物的GC含量一般为40-60%,以45-55%为宜,过高或过低都不利于引发反应。有一些模板本身的GC含量偏低或偏高,导致引物的GC含量不能在上述范围内,这时应尽量使上下游引物的GC 含量以及Tm 值保持接近(上下游引物的GC含量不能相差太大),以有利于退火温度的选择。如果G-C比例超出,则在引物的5’端增加As或Ts;而如果A-T比例过高,则同样在5’端增加Gs或Cs。但也有认为:原来普遍认为PCR引物应当有50%的GC/AT比率的观点其实是不对的,以人基因组DNA为模板,用81%AT的引物可以产生单一的、专一的、长250 bp,含有70% AT的产物。完全没有必要复杂地去计算产物和引物的解链温度,PCR引物的GC/AT比率应当等于或高于所要放大的模板的GC/AT比。产物中GC含量最好大于引物中的GC含量。

二、Degeneracy 多义性

当设计多义引物时应尽量减少引物多义性,这样会带来更好的特异性,应尽量避免3末端的多义性,因为这里即使一个碱基的错配都能阻止引物延伸。

三、3’ End Stability 3 末端稳定性

引物稳定性影响它的错配效率,一条理想的引物应该有一个稳定性较强的5 末端和相对稳定性较弱的3 末端。如果引物3 稳定性强,有可能在即使5 末端不配对的情况下造成错配,形成非特异性扩增条带(secondary bands) 。而3 末端稳定性低的引物较好的原因是在引物发生错配时,由于3 末端不太稳定引物结合不稳定而难以延伸。

四、GC Clamp GC钳

引物与目的位点的有效结合需要有稳定的5 末端。这一段有较强稳定性的5 末端称为GC钳。它保证引物与模板的稳定结合。选择有合适稳定性的引物能在确保不产生非特异性条带的前提下尽量降低退火温度。

五、Secondary Structures二级结构

二级结构是引物设计中必须考虑的一个重要因素。二级结构能显著影响反应中能与模板正确结合的引物数量,发卡结构的存在能限制引物与目的位点的结合能力,从而降低扩增效率,形成发卡环的引物则不能在PCR扩增中发挥作用。

六、Hairpin发卡结构

发卡结构的形成是由于引物自身的互补碱基分子内配对造成引物折叠形成的二级结构,并由于发卡结构的形成是分子内的反应,仅仅需要三个连续碱基配对就可以形成。发卡结构的稳定性可以用自由能衡量。自由能大小取决于碱基配对释放的能量以及折叠DNA形成发卡环所需要的能量,如果自由能值大于0 则该结构不稳定从而不会干扰反应,如果自由能值小于0 则该结构可以干扰反应。

七、Dimer 二聚体

引物之间的配对区域能形成引物二聚体,它是相同或不同的两条引物之间形成的二级结构。它造成引物二聚体扩增并减少目的扩增产物,二聚体可以在序列相同的两条引物或正反向引物之间形成,如果配对区域在3 末端问题会更为严重,3 末端配对很容易引起引物二聚体扩增。

八、False Priming 错配

如果引物可以结合除目的位点外的其他区域,扩增效率将明显降低目的产物带将减少或出现涂布(smear)。3 末端连续几个碱基配对形成错配的倾向要高于引物上游区域同样数量的碱基配对,在使用引物设计软件时,您可以分别设定确认为错配的3 末端或引物全长形成连续碱基配对的数量。

九、自由能问题(如何根据自由能判断引物质量)

1、ΔG值(自由能)反映了引物与模板结合的强弱程度。一般情况下,引物的ΔG值最好呈正弦曲线形状,即5’端和中间ΔG值较高,而3’端ΔG值相对较低,且不要超过9(ΔG值为负值,这里取绝对值),如此则有利于正确引发反应而可防止错误引发。3′末端双链的ΔG是0~-2 kcal/mol时,PCR产量几乎达到百分之百,随着其绝对值的增加产量逐渐下降,在-6时只有40%、到-8时少于20%、而-10时接近于0。

2、引物二聚体及发夹结构的能量一般不要超过4.5,否则容易产生引物二聚体带而且会降低引物浓度从而导致PCR 正常反应不能进行,与二聚体相关的一个参数是碱基的分布,3’端的连续GGG 或CCC 会导致错误引发。二聚体形成的能值越高越稳定,越不符合要求。与二聚体相同,发夹结构的能值越低越好。虽然有些带有发夹环,其ΔG为-3 kcal/mol的自身互补引物也可以得到不错的结果,但是如果它的3′末端被发夹环占据时就很麻烦,即会引发引物内部的延伸反应,减少了参与正式反应引物的数量。当然,如果发夹环在5′末端对反应就没有多大的影响了。

十、产物需要测序的PCR引物的设计问题

在DNA测序的PCR中最好用5′末端稳定(如GC含量较多),而3′末端不太稳定(如AT含量较多)的引物,这种引物的结构可以有效地消除假引发反应。这就是基于引物内部稳定性的经验之谈。其3′末端稳定性低的引物在这些反应中能起好作用的原因在于,接近或在3′末端上的碱基与非靶位点碱基所形成的配对的稳定程度还不足以引发DNA合成,所以不会产生假产物。因此,为了有效地引发反应,引物的5′末端和中央部分必须与靶DNA也形成双链。与此相反,带有稳定的、GC丰富的3′末端的寡核苷酸不需要其所有的核苷酸序列都与靶序列配对,只凭借其3′末端与靶序列任何位点的牢固配合就可以引发反应,产生非专一产物。无论如何,寡核苷酸3′末端最后5个核苷酸的稳定性小于-9 kcal/mol的,通常就是专一性的探针或引物。寡核苷酸3′末端越不稳定,假引发的可能性越低。

顺便说一下,如果新手刚开始接触引物设计,推荐使用Primer Premier 5.0,因为它界面简单,易学易用;如果你想把引物设计得尽善尽美,公认的首选软件是Oligo,其次我认为是DNAstar。Oligo功能强大,所以使用起来就没有Primer Premier 5.0那么简便。先用Primer Premier 5.0设计,然后把设计好的引物拿到Oligo里去检测这对引物的优劣,我想这对大多数引物设计者是一个不错的选择!

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