玉米有丝分裂染色体上玉米BAC探针的FISH杂交体系的构建

Constructing FISH System with Maize BAC Probes on Mitotic Chromosomes of Maize

本研究分别探讨了玉米和水稻基因组c0tDNA对探针的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化对BAC-FISH杂交的影响;探讨了玉米BAC探针中重复序列含量对FISH信号的影响.初步形成了一套以玉米BAC探针在玉米有丝分裂染色体上进行FISH杂交的优化技术体系.结果表明,玉米基因组c0tDNA对探针封阻的c0t值应小于50;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻基因组的c0t-100DNA封阻探针重复序列对BAC-FISH杂交信号特异性的改善具有明显的效果;同时,验证了选择重复序列含量较少的玉米BAC作为FISH杂交的探针也是获得特异性杂交信号的重要条件.

Fluorescence in situ hybridization （FISH） using the maize BAC-DNA as a probe had been performed on mitotic chromosomes. To optimize the BAC-FISH technique, we respective investigated signal specificities of FISH by analyzing the maize genomic Co t DNA and rice genomic Co t DNA blocking repetitive sequences of the probes-BAC, washing stringency after hybridization, FAD concentration in hybridization mixtures and containing repetitive sequence of the maize probes-BAC. The results showed that Co t DNA of maize genomic blocked the repetitive sequence of the probes when the Co t value was below 50. Meanwhiles, decreasing the FAD concentration in hybridization mixtures, adjusting the washing stringency after hybridization, especially blocking the repetitive sequences of maize probes-BAC with rice genomic Co t-100 DNA which could improve the signals of BAC-FISH. The results demonstrated a important factor of receiving specific signals of BAC-FISH by select maize BAC-probes for lesser content repeat sequences.