硅对PCR过程的抑制作用研究

Studies of Silicon Inhibition Effects on Polymerase Chain Reaction

探讨不同氧化程度的硅材料对PCR扩增的抑制作用及其机理。将不同氧化程度的硅纳米颗粒加入PCR反应液中 ,使其与Taq酶、模板等充分接触 ,通过离心将硅纳米颗粒沉降在管壁上 ,取出上清或保留硅纳米颗粒上机扩增 ,扩增产物采用凝胶电泳法检测。结果表明 ,随着硅材料表面面积与PCR反应液体积之比的增大 ,核酸扩增效率将明显下降 ,并且在所研究的范围内 ,氧化程度高的硅材料对PCR过程抑制作用更强 ;通过对抑制作用机理进行初步的实验研究 ,表明硅材料对PCR反应液中的Taq酶的吸附是导致抑制现象产生的主要原因 ,而对模板的吸附影响较小 ;并且 ,反应管内是否保留硅材料对核酸扩增影响较小 ,硅材料没有明显的直接化学抑制作用。

Polymerase chain reaction (PCR),thermally activated chemical reactions for nucleic acid amplifications,is one of the most important molecular biological methods.With the development of silicon microfabrication techniques,silicon based micro PCR chips have been widely studied to achieve rapid PCR as well as cutting down the power consumption and the required space.Although silicon offers many substantial advantages in establishing PCR instruments,it has been repeatedly reported that silicon related materials are not compatible with the polymerase chain reaction.Silicon inhibition effects on the polymerase chain reaction were experimentally investigated and the inhibition mechanism was tentatively explored.Two kinds of silicon nanoparticles with different oxidation degrees were added into PCR mixtures to activate possible reactions between the silicon related materials and the bio reagents.By high speed centrifuging,the nanoparticles subsided on the tube inner surface.Extracting the supernatant or remaining the silicon nanoparticles inside the PCR mixture for the following nucleic acids amplification,and the amplification results were detected by the agarose gels electrophoresis.The experimental results demonstrated that with the surface to volume ratio (the ratio of the silicon surface area to the PCR mixture volume) increasing,the amplification efficiencies decreased considerably,and within the studied ranges,the higher surface oxidation degree,the stronger silicon inhibition effects on the PCR was.And the experimental results also indicated that the silicon inhibition effects should be attributed to the adsorption of Taq polymerase on the silicon related material surface,and the proof that the silicon related material directly participates in the amplification process is not available.