Study On The Mechanical Properties Of Braiding-DNA And Side-view And High-throughput Single Molecule Techniques

With the deep understanding and research,it has been found that the mechanical properties of DNA play a vital role in the biological processes such as DNA replication,transcription and recombination.Over the past decades,many studies have been done on the elastic properties of single DNA molecules.Through the measurement of the force and extension of a single DNA molecule,the mechanical properties of the molecule is allowed to directly determined,the polymer elasticity theory is proved,the structural transformation caused by mechanical stress is revealed,and the experimental and conceptual framework for the mechanical experiment of enzymes that act on DNA is established.However,the situation of DNA molecules in the cell is more complex,we need to study the mechanical properties of braiding-DNA or even multiple-DNA molecules.And in recent years,the development of single-molecule manipulation and detection technology provide the necessary technical support for our research.Here we study DNA braiding using a magnetic tweezers system.The two ends of the experimental molecules were fixed on the slide and a magnetic microbead,and then add a magnetic field to control the magnetic microbead,so that the biological molecules can be stretched or twisted.Through the change of magnetic microbead position,we can calculate the length of the bio-molecule,the force and the torque.In the experiment,we measured the extension of DNA under different force and calibrated the ideal braided-DNA molecule by the worm chain model.Accord the Maxwell method to calculate the relative torque of braided-DNA molecules.Experiments show that the braided-DNA molecules in the introduction of one turn(|n|=1),the length will be significant reduced,when |n|>1,the change is more gentle.The torque is affected by the externally applied force,the number of revolutions and the molecular spacing.Under the same external force,the torque increases with the number of catenation.At the same catenation number,the torque increases with the force.The distance between the two DNA molecules also has some influence on the torque.At the same force and the same catenation number,the greater spacing between the two DNA molecules,the greater torque.The general magnetic tweezers system can only manipulate the DNA molecules.On this basis,we design a dual imaging magnetic tweezers system,which can carry out the fluorescence signal detection while manipulating the DNA molecules.Using dual optical path in both horizontal and vertical direction to observe biomolecules and cell samples.The resulting structural and functional information is checked against each other,reflecting changes in the three-dimensional space over time and the results are more convincing.Combined with the advantages of micro-nano-scale force spectrum and spectroscopy,we can visually detect the length of individual cells or biomolecules and the force applied,and observe the images.As with other single-molecule techniques,magnetic tweezers can only study one molecule at a time.To improve detection efficiency,we have improved a multi-channel optical inspection system.Each reaction chamber in the multichannel reaction chamber corresponds to a single channel,and each individual channel detects a single molecule level reaction,so that the fluorescence generated by the plurality of reaction chambers does not overlap with each other.The positive and negative contrast in the image is so clear that when the analyte is a trace the results of the multiple reaction chambers are accurate and stable.This system adds a quenching device to improve the detection accuracy of the system.