Max-Planck-Institute for Polymer Research, D-55128 Mainz, Germany.
Biointerphases. 2006 Dec;1(4):113-22. doi: 10.1116/1.2365386.
Peptide nucleic acid (PNA), a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA/DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin/biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association (k(on)) and dissociation processes (k(off)), and the affinity constant (K(A)) of the PNA/DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA (15mer) target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product (169mer) obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength.
肽核酸（PNA）是一种聚酰胺 DNA 类似物，由于 PNA/DNA 双链的稳定性，它激发了各种基于杂交的方法的发展，用于核酸的检测、定量、纯化和表征。在这项工作中，通过使用链霉亲和素/生物素结合将与 Roundup Ready 大豆特定序列互补的 PNA 探针固定到传感器表面上的自组装基质上。通过使用表面等离子体场增强荧光光谱术实时将标记有发色团的 DNA 靶标寡核苷酸施加到 PNA 修饰的 Au 传感器表面上，监测 PNA 和 DNA 的特异性杂交。作者通过将数据拟合到简单的 Langmuir 模型，展示了三种实验，称为全局、滴定和动力学分析，用于确定 PNA/DNA 双链形成的缔合（k(on))和离解过程（k(off))以及亲和常数（K(A))。记录了在 15mer PNA 探针上对单个碱基错配 DNA（15mer）靶标的区分，其亲和常数相差两个数量级。最后，通过动力学滴定分析确定了从遗传修饰大豆参考材料中提取的 DNA 扩增得到的长聚合酶链反应产物（169mer）杂交的亲和常数。结果表明，即使在低离子强度下与 PNA 杂交，高靶标表面覆盖率也会产生库仑势垒的影响。
