Fluorescence in situ detection of short DNA sequences

描述（由申请人提供）：提出了一种用于DNA荧光原位检测的全新方法，该方法可以在非变性条件下检测中期染色体扩散和间期细胞核中的短（约20 bp长）单拷贝DNA序列。待开发的短序列荧光原位检测方法（FISDOSS）将具有极高的特异性，因为环状探针将通过将合成寡核苷酸连接到由专门设计的肽核酸（PNA）打开的短DNA序列上来组装。高灵敏度将提供一个有效的污染免疫等温信号放大方法：滚环扩增（RCA）的组装的环状探针与许多荧光标记的核苷酸的掺入。所有程序将直接在载玻片上进行，间期核和中期染色体的最终检测将通过标准技术使用荧光显微镜进行。在第一阶段，将在任意选择的人类基因组独特位点上进行原理验证实验。第一阶段的目标是证明，在初始优化后，可以在非变性中期人类染色体内有效和特异性地检测短特异性序列。该方法将被扩展到平行多个检测的各种独特的网站在人类基因组中。为了证明FISDOSS适用于检测癌症的遗传标志物，将检测12个与慢性淋巴细胞白血病（CLL）相关的适当位点。该项目的实施将产生一种方便的荧光原位技术，具有在DNA水平上进行可靠和高度灵敏的癌症诊断的巨大潜力。

DESCRIPTION (provided by applicant): A radically new approach for the fluorescence in situ detection of DNA is proposed, which makes it possible to detect short (about 20-bp-long) single-copy DNA sequences in metaphase chromosome spreads and in interphase nuclei under non-denaturing conditions. The method of fluorescence in situ detection of short sequences (FISDOSS) to be developed will be exceedingly specific because a circular probe will be assembled via ligation of synthetic oligonucleotides on short DNA sequences opened up by specially designed peptide nucleic acids (PNAs). A high sensitivity will be provided by an efficient contamination-immune isothermal method of signal amplification: rolling-circle amplification (RCA) of the assembled circular probes with incorporation of numerous fluorescently labeled nucleotides. All procedures will be performed directly on slides and the final detection of interphase nuclei and metaphase chromosomes will be done by standard techniques using a fluorescent microscope. In Phase I, proof-of-principle experiments will be performed on arbitrarily chosen sites unique for the human genome. The goal of Phase I is to demonstrate, after initial optimization, that the short specific sequences can be effectively and specifically detected within non-denatured metaphase human chromosomes. The method will be extended to parallel multiple detection of various unique sites in the human genome. To demonstrate that FISDOSS is applicable to detect genetic markers of cancer, 12 appropriate sites associated with chronic lymphocytic leukemia (CLL) will be tested. The implementation of the project will yield a convenient fluorescent in situ technique with a great potential for reliable and highly sensitive diagnosis of cancer on the DNA level.