Ultra-specific In Situ Detection of Short Sequences in Human Genomic DNA under No

描述（由申请人提供）：该项目的目标包括开发一种全新的技术，用于以天然双链形式检测基因组DNA中的DNA标记位点。这种新方法是基于肽核酸（PNA）在双链DNA中打开独特短序列（20-30bp）的能力，使得在非变性条件下检测基因组DNA中的短DNA序列成为可能。在一对PNA开子选择性打开的位点上，组装一个环状寡核苷酸探针，作为滚动环扩增（RCA）的模板。待开发的方法的主要优点在于其巨大的特殊性，这包括两个主要特点。首先，使用非常短的PNA低聚物，这导致对DNA结合位点的错配零容忍。其次，只有被PNA开子打开的位点才能被环状寡核苷酸结合，这需要超越环状探针形成的特异性。该项目的可行性基于PI最近公布的数据，其中类似的方法已成功实施，用于开发一种新的细菌检测方法。在该项目中，这些研究将扩展到检测真核细胞（包括人类细胞）中双链基因组DNA内选定的DNA靶点。虽然将这种方法应用于真核细胞比应用于细菌要困难得多，但人类细胞核的初步数据非常有希望。即将开发的方法将能够以一种方便的类似fish的形式，在人类染色体上原位定位和具体标记单个基因，以检测染色体中的各种插入和转位，包括与疾病相关的基因组不稳定性。随着项目的实施，将开发出在自然条件下标记和检测dsDNA的强大方法，这些方法在病理学上具有潜在的广泛应用前景，这将为DNA诊断开辟新的途径。

DESCRIPTION (provided by applicant): The goal of the project consists in the development of a radically new technology for specific detection of DNA signature sites within genomic DNA in its native, double-stranded form. The new approach is based on the ability of peptide nucleic acid (PNA) to open up unique short sequences (20-30bp) within duplex DNA and make it possible to detect short DNA sequences within genomic DNA under non-denaturing conditions. On the site selectively opened by a pair of PNA openers, a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). The principal advantage of the approach to be developed consists in the enormous specificity, which entails two its major features. First, very short PNA oligomers are used, which result in zero tolerance to mismatches in the sites of DNA binding. Secondly, only sites opened by PNA openers are accessible to binding by circularazible oligonucleotides, which entails exceeding specificity of circular probe formation. The feasibility of this project is based on the data recently published by PI, in which similar approach has been successfully implemented for the development of a new method for bacterial detection. In the project these studies will be extended to detect chosen DNA target sites within double-stranded genomic DNA in eukaryotic cells including human cells. While adaptation of the approach to eukaryotic cells is expected to be much more challenging than in case of bacteria, the preliminary data on human nuclei are very promising. The approach to be developed will make it possible to target and specifically label individual genes on human chromosomes in situ, to detect various insertions and transpositions in chromosomes, including diseases-associated genomic instabilities, in a convenient FISH-like format. As a result of the project implementation, robust approaches for marking and detection of dsDNA under native conditions with potential wide applications in pathology will be developed, which will open new avenues in DNA diagnostics. Public Health Relevance Statement: The project's goal consists in development of a rapid, exceedingly specific and very sensitive approach for specific recognition, marking and localization of short DNA signature sites within genomic DNA in its native, double-stranded form. Its implementation carries great promise to equip biomedical and clinical researchers with new powerful tools for quick and reliable diagnosis. This diagnostic will be capable to target and specifically label individual genes on human chromosomes in situ, to detect various insertions and transpositions in chromosomes, including diseases-associated genomic instabilities, in a convenient FISH-like format.