Fluorescence in situ detection of short DNA sequences

描述（由申请人提供）：提出了一种全新的DNA荧光原位检测方法，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。短序列荧光原位检测（FISDOSS）的方法将是非常特殊的，因为一个圆形探针将通过连接合成的寡核苷酸在由特殊设计的肽核酸（PNAs）打开的短DNA序列上组装。一个高灵敏度将提供一个有效的污染免疫等温信号扩增方法：滚动圈扩增（RCA）组装的圆形探针与大量荧光标记核苷酸的掺入。所有程序将直接在载玻片上进行，间期细胞核和中期染色体的最终检测将使用荧光显微镜通过标准技术完成。在第一阶段，原理验证实验将在任意选择的人类基因组独特的位点上进行。第一阶段的目标是在初始优化后证明，短特异性序列可以在非变性中期人类染色体中有效和特异性地检测到。该方法将扩展到对人类基因组中各种独特位点的并行多重检测。为了证明FISDOSS适用于检测癌症的遗传标记，将测试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水平上可靠和高灵敏度诊断癌症的巨大潜力。

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.