Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation
Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation
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DOI：
10.1111/j.1365-313x.2009.03881.x
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发表时间：
2009-08-01
期刊：
影响因子：
7.2
通讯作者：
Jiang, Jiming
中科院分区：
文献类型：
--
作者：
Koo, Dal-Hoe;Jiang, Jiming
基于减数分裂粗线期染色体的荧光原位杂交（FISH）技术是植物分子细胞遗传学研究的重要工具之一。在这里，我们报告了一个简单的技术，使玉米粗线期染色体拉伸到至少20倍的原始大小。在该过程中使用了改良的Carnoy II固定剂（6：1：3乙醇：氯仿：乙酸），并证明是粗线期染色体超拉伸的关键。我们证明，超拉伸粗线期染色体提供了前所未有的分辨率为染色体为基础的FISH映射。DNA探针分离少至50 kb，可以解决超拉伸染色体。FISH与超伸展粗线期染色体上5-甲基胞嘧啶的免疫荧光检测相结合，提供了一种强有力的工具来揭示特定染色体结构域的DNA甲基化，特别是那些与高度重复DNA序列相关的结构域。
P>Meiotic pachytene chromosome-based fluorescence in situ hybridization (FISH) mapping is one of the most important tools in plant molecular cytogenetic research. Here we report a simple technique that allows stretching of pachytene chromosomes of maize to up to at least 20 times their original size. A modified Carnoy's II fixative (6:1:3 ethanol:chloroform:acetic acid) was used in the procedure, and proved to be key for super-stretching of pachytene chromosomes. We demonstrate that super-stretched pachytene chromosomes provide unprecedented resolution for chromosome-based FISH mapping. DNA probes separated by as little as 50 kb can be resolved on super-stretched chromosomes. A combination of FISH with immunofluorescent detection of 5-methyl cytosine on super-stretched pachytene chromosomes provides a powerful tool to reveal DNA methylation of specific chromosomal domains, especially those associated with highly repetitive DNA sequences.