High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes
High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes
复制标题
使用花青和荧光素染料进行高分辨率多色荧光原位杂交：通过直接荧光标记的阿尔菲德 DNA 探针快速鉴定染色体
作者：
Y. Yurov;I. V. Soloviev;S. Vorsanova;B. Marçais;Gerard Roize;R. Lewis
我们测试了直接用荧光标记核苷酸（Cy3-dCTP、Cy5-dCTP、FluorX-dCTP）标记的 DNA 探针，用于人类染色体的高分辨率单色和多色检测以及通过原位杂交分析着丝粒 DNA 组织。特异性针对1、2、3、4+9、5+19、6、7、8、10、11、13+21、14+22、15、16、17、18、20、22、X和Y染色体的α卫星DNA探针适用于准确鉴定中期和间期细胞中的人类染色体。 Cy3标记探针具有以下几个优点：（1）荧光水平高（是荧光素标记探针的5-10倍）； (2)溶液中荧光水平低，允许在杂交过程中原位检测目标染色体，无需清洗载玻片； (3)在长时间（1-2小时）暴露于强光下时具有高抗光漂白性，从而允许在数字成像显微镜的图像采集过程中使用高能汞灯或较长的积分时间来确定微弱信号。对于双色和多色荧光原位杂交 (FISH)，我们成功地使用了直接荧光探针的不同组合，并通过传统显微镜或数字成像显微镜保存图像。 FluorX 和 Cy3 染料允许使用粘粒探针在一步杂交实验中进行绘图。花青标记的荧光 DNA 探针为在简单的 15 分钟 FISH 过程中快速染色体检测提供了额外的可能性，并且可以推荐用于利用 FISH 的基础研究和临床研究。
We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5–10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH.
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DOI：
10.1073/pnas.83.9.2934
发表时间：
1986-05-01
影响因子：
11.1
作者：
PINKEL, D;STRAUME, T;GRAY, JW
通讯作者：
GRAY, JW
影响因子：
4.7
作者：
MUJUMDAR, RB;ERNST, LA;WAGGONER, AS
通讯作者：
WAGGONER, AS
影响因子：
3.5
作者：
Ried, Thomas;Landes, Greg;Ward, David C.
通讯作者：
Ward, David C.
DOI：
10.1073/pnas.89.4.1388
发表时间：
1992-02-15
影响因子：
11.1
作者：
RIED, T;BALDINI, A;WARD, DC
通讯作者：
WARD, DC