Reddy Banda Suresh, Klapproth Holger, Smit Nicolaas, Bednar Sonja, Brandstetter Thomas, Rühe Jürgen
Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany.
Safeguard Biosystems Holding Ltd., London, United Kingdom.
Front Bioeng Biotechnol. 2022 Nov 30;10:1045154. doi: 10.3389/fbioe.2022.1045154. eCollection 2022.
The sensitivity of a PCR based biochip assay relies on the efficiency of PCR amplicons in binding to the microarray spots. The essential factor determining the sensitivity is the amount of single stranded (ss) amplicons available for biochip hybridization. Asymmetric PCR can generate ss-amplicons depending on the ratio of primers used in the amplification process, but this process is often inefficient. We report a novel variant of PCR called the Asymmetric Exponential and Linear Amplification (AELA) which can overcome these issues and generate large amounts of single stranded amplicons. AELA-PCR introduces an amplification strategy that makes use of both exponential and linear amplification of the target nucleic acid. This is done by specifically designed primers and choice of adequate thermal profiles. In conventional PCR with a classical thermal profile, these specifically designed primers will work normally and contribute to an exponential increase of amplicons. A designed sequence extension of one of the primers and a very specific thermal profile, will result in a situation that the extended primer will be the only functional one for amplification, resulting in a linear phase of the amplification process. That is why during this step only one of the two strands of the target is amplified linearly and no longer exponentially. The result of the whole process is an amplification product enriched very strongly in one of the two single strands of the target. These adaptions in PCR are particularly favorable where the generation of ss-DNA/RNA is required. We demonstrate the higher biochip sensitivity of AELA-PCR compared to conventional amplification methods with an example of the detection on a DNA oligonucleotide microarray.
基于PCR的生物芯片检测的灵敏度取决于PCR扩增产物与微阵列斑点结合的效率。决定灵敏度的关键因素是可用于生物芯片杂交的单链（ss）扩增产物的量。不对称PCR可以根据扩增过程中使用的引物比例产生单链扩增产物，但这个过程通常效率不高。我们报道了一种名为不对称指数和线性扩增（AELA）的新型PCR变体，它可以克服这些问题并产生大量单链扩增产物。AELA-PCR引入了一种利用目标核酸的指数和线性扩增的扩增策略。这是通过专门设计的引物和选择合适的热循环参数来实现的。在具有经典热循环参数的传统PCR中，这些专门设计的引物将正常工作并导致扩增产物呈指数增加。对其中一个引物进行设计的序列延伸和非常特定的热循环参数，将导致这样一种情况：延伸后的引物将是唯一用于扩增的功能性引物，从而导致扩增过程进入线性阶段。这就是为什么在这一步中，目标的两条链中只有一条被线性扩增而不再是指数扩增。整个过程的结果是扩增产物在目标的两条单链中的一条中高度富集。在需要生成ss-DNA/RNA的情况下，PCR中的这些调整特别有利。我们以在DNA寡核苷酸微阵列上的检测为例，证明了AELA-PCR与传统扩增方法相比具有更高的生物芯片灵敏度。
