Colorimetric DNA Detection Based On Hairpin Assembly Reaction And Construction Of Recognizing Molecular Gene Library

1、 Simple colorimetric DNA detection based on hairpin assembly reaction andtarget-catalytic circuits for signal amplificationDNA is the genetic factor in organisms. The detection of DNA is often connectedto the diagnosis of different disease, so the detection of DNA is very meaningful tostudy. Because of the progress of detection technology for DNA, human have changedthe detection of other biological molecules to the detection of DNA, for exampleprotein and small molecular substances. There are many methods of DNA detection,for example: fluorescence detection method, chemiluminescence detection,colorimetry detection, electrochemical detection and fluorescence quantitative PCR,and so on. In the method of biochemical analysis, the hot issue is increasing thesensitivity of detection, so the technology of signal amplification is widely used. Thetool enzyme-based signal amplification and gold nanoparticle biological bar code arethe hot issue of study. Our topic is based on biological compatibility of goldnanoparticle, binding hairpin DNA probes onto the gold nanoparticle to realize thesignal amplification, and finally detect the target DNA by gold nanoparticlecolorimetry.A simple strategy of colorimetric DNA detection was presented in the presentwork, which was based on hairpin assembly reaction and target-catalytic DNA circuitsto realize enzyme-free signal amplification. The method employed two hairpin speciesH1and H2, which were stable and unable to hybridize in the absence of target. In the presence of target, the target hybridized with hairpin H1and the opened hairpin H1hybridized with hairpin H2, which made the target be displaced. The duplex of hairpinH1and H2modified on nanoparticles made particle aggregation be visualized with thenaked eyes. The displaced target again triggered the next round of strand-exchangereaction to realize signal amplification. The method would have a wide range ofsensing applications with enzyme-free, easy operation and extended field ofapplication.Our approach is simple, sensitive, and selective. This approach could be appliedto a wide range of sensing applications without enzyme, easy operation and extendedfield of application. The method could detect as little as2.2nM target DNA and ishighly selective for completely complementary DNA. The approach would also beused to detect extensive targets including proteins, small molecules, and metal ionsthat can produce initiator DNA strand. In addition, this approach may be a potentiallyuseful tool in the fields of basic and clinical research, and diagnostics.2、 Analysis and design of structure domain and preliminary optimization ofrecombination for geneBiological molecular recognition element is an important part of biological sensor.Looking for a variety of biological molecular recognition element is one of theimportant development directions of biological sensor. In this part, we mainly used thecombined gene modules of directed evolution methods to construct the diversity ofgene library, searching for novel protein recognition molecule.For convenience of screening, this experiment took kanamycin resistance geneprotein as recognition molecules to verify the identification of library constructionmethod was effective. As the kanamycin resistance gene a example，we explored theprocess of directed evolution. First of all, we looked up the kanamycin resistance genefrom GeneBank and analyzed the gene sequence. According to the two-dimensionalstructure we designed the DNA segment to carry through the reassembly PCR. Theconstruction of this method will play a positive role for detection of target that is hardto identify, for example，screening of new target receptor and prevention and treatmentof the newly discovered virus.