Musheev Michael U, Krylov Sergey N
Department of Chemistry, York University, Toronto, Ont., Canada M3J 1P3.
Anal Chim Acta. 2006 Mar 30;564(1):91-6. doi: 10.1016/j.aca.2005.09.069. Epub 2005 Nov 2.
Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.
适配体是能够以高亲和力和选择性结合不同类型靶标的DNA寡核苷酸。作为蛋白质多重定量分析中的亲和探针，它们特别具有吸引力。适配体通常从随机DNA序列的大型文库中通过一种称为指数富集配体系统进化（SELEX）的通用方法进行筛选。SELEX包括两个过程的重复轮次：（i）通过亲和方法将适配体与非适配体分离，以及（ii）通过聚合酶链反应（PCR）扩增适配体。最近引入了具有极高分离效率的新分离方法。为了使整个SELEX程序高效，新分离方法的高效率必须与PCR的高效率相匹配。在此，我们首次对适配体筛选中使用的随机DNA文库的PCR扩增进行了详细研究。以毛细管电泳作为分析工具，我们发现了均匀DNA模板的PCR扩增与随机DNA序列大型文库的PCR扩增之间的根本差异。均匀DNA模板的产物形成一直持续到引物耗尽。对于以随机DNA文库为模板的情况，当PCR引物仍过量于产物时，产物积累就会停止。然后产物迅速转化为副产物，并且在仅额外进行5个PCR循环后几乎消失。产物的产量随着文库中DNA分子长度的增加而降低。我们还证明了PCR混合物中DNA分子的初始数量对副产物的形成没有影响。而PCR混合物中Taq DNA聚合酶浓度的增加会选择性地提高PCR产物的产量。我们的研究结果表明，均匀DNA样品的PCR扩增标准程序不能直接应用于随机DNA文库的PCR扩增：为确保高效的SELEX，必须针对随机DNA文库优化PCR扩增。
