Single-primer-limited amplification: A method to generate random single-stranded DNA sub-library for aptamer selection
Single-primer-limited amplification: A method to generate random single-stranded DNA sub-library for aptamer selection
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单引物有限扩增：一种随机生成单链 DNA 子文库用于适体选择的方法
DOI：
10.1016/j.ab.2013.05.008
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发表时间：
2013-09-01
影响因子：
2.9
通讯作者：
Lv, Nong-Hua
中科院分区：
文献类型：
--
作者：
He, Chao-Zhu;Zhang, Kun-He;Lv, Nong-Hua
通过聚合酶链式反应（PCR）扩增随机单链DNA（ssDNA）文库是每轮通过指数富集配体系统进化（SELEX）选择适体的关键步骤，但由于PCR系统中各种序列之间存在严重的非特异性杂交，副产物的扩增可能会阻碍其扩增。为了扩增无副产物的随机单链DNA文库，我们开发了一种称为单引物有限扩增（SPLA）的新方法，该方法首先使用限制为模板摩尔量5倍的反向引物扩增单链DNA文库的负链DNA（msDNA），然后使用限制为模板摩尔量10倍的正向引物扩增msDNA的正链DNA（psDNA）并回收psDNA 通过凝胶切除。我们发现副产物的量随着模板用量和热循环次数的增加而增加。通过优化的模板量和热循环，SPLA 可以扩增目标 ssDNA，而不会检测到副产物和非特异性产物，并且产生的 psDNA 产量是不对称 PCR 的 16.1 倍。总之，SPLA是一种简单可行的方法，可以有效地生成用于适体选择的随机ssDNA子库。 (C) 2013 Elsevier Inc. 保留所有权利。
The amplification of a random single-stranded DNA (ssDNA) library by polymerase chain reaction (PCR) is a key step in each round of aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX), but it can be impeded by the amplification of by-products due to the severely nonspecific hybridizations among various sequences in the PCR system. To amplify a random ssDNA library free from by-products, we developed a novel method termed single-primer-limited amplification (SPLA), which was initiated from the amplification of minus-stranded DNA (msDNA) of an ssDNA library with reverse primer limited to 5-fold molar quantity of the template, followed by the amplification of plus-stranded DNA (psDNA) of the msDNA with forward primer limited to 10-fold molar quantity of the template and recovery of psDNA by gel excision. We found that the amount of by-products increased with the increase of template amount and thermal cycle number. With the optimized template amount and thermal cycle, SPLA could amplify target ssDNA without detectable by-products and nonspecific products and could produce psDNA 16.1 times as much as that by asymmetric PCR. In conclusion, SPLA is a simple and feasible method to efficiently generate a random ssDNA sub-library for aptamer selection. (C) 2013 Elsevier Inc. All rights reserved.