Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Biochem Biophys Res Commun. 2012 Jan 6;417(1):404-8. doi: 10.1016/j.bbrc.2011.11.128. Epub 2011 Dec 7.
Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
基因组 DNA 的光学作图在许多应用中都具有重要意义，例如测序的支架构建以及结构变异的检测，以及细菌和病毒等病原体的检测。对于未来的临床应用，最好有一种基于尽可能少的步骤的快速而稳健的作图方法。我们在这里展示了一种一步法，可获得直接使用纳米流体装置和荧光显微镜观察的 DNA 条码。使用 YOYO-1 混合物（一种明亮的 DNA 染料）和 netropsin（一种具有非常高 AT 特异性的天然抗生素），我们获得了沿 DNA 的荧光强度分布的 DNA 图谱，该图谱反映了潜在的序列。netropsin 与 AT-四联体结合，并阻止这些结合位点与 YOYO-1 结合，从而导致 DNA 中富含 AT 的区域的荧光强度降低。因此，我们获得了一种 DNA 条码，其富含 AT 的区域较暗，富含 GC 的区域较亮，具有千碱基分辨率。我们通过在噬菌体 T4 的 DNA 上获得条码来证明该方法的多功能性，该条码捕获了其环状排列，并且与已知序列非常吻合。
