Applications Of Sensitive Nucleic Acids Amplification In Biosensors

Small molecular-protien interaction and copy number variations (CNVs) are always important analytes as they are closely concerned with human beings’work and livelihood. Proteins are important biomolecules in the field of biochemistry and life science. Proteins can express physiological characters directly. They are useful to sustain the metabolism within the organism and they are also the carrier of many physiological functions. It is significant for researchers to disclose the secrets of life and develop new medicine to conquer many chronic diseases. What we should do is to further study the interaction mechanism between small molecules and proteins. Moreover, copy number variations are crucial to research genome evolutions and phenotype differences, thus it is recognized as a reason for individual differences in genome sequence. Many scientific researches show that CNVs can result in different levels of gene expression and are connected with the normal phenotypic variation and human diseases. Hence, constructing simple and rapid methods for the study of small molecules-protein interaction and copy number variations is significant in biological fields. Techniques of nucleic acid amplification hold great potential in biosensor technology as they are endowed with numerous advantages such as sensitive, specific and time-saving and so on. In this paper, we have developed several detection methods for small molecules-protein interaction and copy number variations based on nucleic acid amplification methods as follows:(1) We developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. The results revealed that the small molecule-protein interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Three small molecule compounds and their protein receptors have been used to demonstrate the generalization of the developed strategy.(2) We invented a method to detect the copy number of given region from genome sequence at the same time by employing theα-globin gene as the material for testing, basing on the nested multiplex real-time qPCR system. We used three pairs of target capture probes to amplify targeted DNA sequences. The amplification productions included specific genome sequence, hydrolysis sequence to capture fluorescent probes and universal sequence to hybridize with universal primers. The production was used as the template for the second amplification, which was extended under the help of univeral primers and then combined with different fluorescent probes. The Cq values of different genes were obtained after all theses steps. Our method can detect the copy number variations like deletion and replication inα-globin gene.