Abstract：
DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end\'s length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.
摘要：
在固体支持物上发生的DNA杂交现象不能像水相杂交那样清楚地理解,并且数学模型不能预测一些凭经验获得的结果。正在进行的研究已经确定了重要的参数，但仍然不完整，无法准确解释所有相互作用。先前已经显示,在与捕获探针杂交后,靶DNA链的突出(悬空)末端的长度与与溶液中的互补链的相互作用相关,这可以导致靶的脱离结合及其从表面的释放。我们已经开发了一种仪器，用于实时监测用寡核苷酸捕获探针功能化的球形颗粒上的DNA杂交，并在微流体盒内以紧密堆积的单层珠床的形式排列。该仪器配备有气动模块以调节药筒上的流体的移位。我们将该系统与载玻片上的常规（被动）和离心驱动（主动）微流体微阵列杂交进行了比较，以建立检测单核苷酸多态性的性能水平。该系统还用于实时研究当固定的靶DNA暴露于溶液中的互补链时悬空末端长度的影响。我们的发现表明，增加悬空末端的长度会导致目标扩增子以接近初始杂交过程的速率从与珠子结合的捕获探针中解吸。最后,使用从随机临床样品中获得的无乳链球菌cfb基因扩增子进行珠床杂交，允许在5-15分钟内鉴定B组链球菌。本文提出的方法可用于研究固体支持物上的竞争性杂交机制，并快速验证微阵列捕获探针的适用性。
