서강대학교

초록/요약

Full stretching of single genomic DNA molecules has received great attention for investigation in biophysical and biochemical studies for recent years. Since this critical step provides the platform of analysis of genomic DNA with high resolution, it is important to develop more efficient and simple method elongating DNA molecules. Here, we present two methods for sufficiently elongating single genomic DNA molecules and its applications. The first method relies on conditions of nanochannel dimension and ionic strength that a DNA molecule can stretch almost its full contour length: the average stretch is 19.1 μm ± 1.1 μm for YOYO-1 stained DNA (21.8 μm contour length) in 250 nm x 400 nm channel, which is the longest stretch value ever reported in any nanochannels or nanoslits. In addition, based on Odijk’s polymer physics theory, we interpret our experimental findings as a function of channel dimensions and ionic strengths. The second platform is constructed by biotin-binding proteins on which DNA molecules can be tethered on one end by ligase reaction and then fully stretched by continuous buffer flow. The strong point of the system is that the tethered DNA molecules can be remained on fixed position with serial different enzyme flows. Moreover, we demonstrate its biocompatibility and efficiency, showing reversible immobilization of genomic DNA by pH control. Upon the immobilized T4 DNA molecules, we show restriction mapping by BbvC1 that prove its circular permutation. This approach for stretching and immobilizing of single genomic DNA molecules has potential to investigate specific loci or gain sequence information.